KR20240016287A - Conjugates of antibodies and functional substances or salts thereof, and compounds or salts thereof used in their production - Google Patents

Abstract

The present invention provides a conjugate of an antibody and a functional material or a salt thereof having excellent desired properties while controlling the binding ratio between the antibody and the functional material within a desired range. More specifically, the present invention relates to the formula (I):
[Formula (I)]

〔During the meal,
Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and also forms an amide bond with a carbonyl group adjacent to Ig regioselectively through the amino group in the side chain of the lysine residue in the two heavy chains,
L ₁ and L ₂ each represent a divalent group,
R ₁ represents a monovalent group that may contain a hydrophilic group,
X shows a good two -stage period even if you have a substituent, wherein the secondary group is 1 to 3, which constitutes the main chain that connects the two atoms on both sides of X, and even if the substituent contains a hydrophilic group. Good monovalent energy,
D represents a functional material,
R _A represents the side chain of the valine residue,
R _B represents the side chain of a citrulline residue or an alanine residue,
n is 0 or 1,
The average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5. A conjugate or salt thereof of an antibody and a functional substance, comprising a structural unit represented by <RTI ID=0.0>and</RTI> and at least one hydrophilic group is present in the structural unit. provides.

Description

Conjugates of antibodies and functional substances or salts thereof, and compounds or salts thereof used in their production

The present invention relates to conjugates of antibodies and functional substances or salts thereof, and compounds or salts thereof used in their production.

Recently, research and development of antibody drug conjugates (ADC) is in full swing. As its name suggests, ADC is a drug that conjugates a drug (e.g., anticancer agent) to an antibody, and has direct cell killing activity against cancer cells, etc. A representative ADC is T-DM1 (brand name: Cadocyra (registered trademark)) jointly developed by Immunogene and Roche.

ADC is produced by binding a functional group in the side chain of a specific amino acid residue present in an antibody to a drug. An example of such a functional group used in the production of ADC is an amino group in the side chain of a lysine residue present in an antibody. As a technique for site-selectively modifying the lysine group (e.g., lysine residue at position 246/248, 288/290, or 317) in an antibody, several techniques have been reported (e.g., Patent Documents 1 to 4).

In ADC, the antibody and drug are connected through a linker. There are various linkers in ADC. For example, in ADC used as an anticancer drug, a peptide made of valine-citrulline (Val-Cit: VC) is used as a linker that is stable in human plasma and has a structure that can be cleaved by a specific enzyme to release the drug within cancer cells. There is a linker containing the structure. The linker containing this dipeptide is stable in human plasma, as shown in (A) below, but the VC structure is recognized by cathepsin B in lysosomes in human cancer cells, as shown in (B) below. , the amide bond present at the carboxy terminal side of citrulline is severed. Therefore, an ADC having a linker containing such a dipeptide can release the drug within human cancer cells and exhibit drug efficacy.

However, ADC having a linker containing the above dipeptide is unstable in mouse plasma (Non-patent Documents 1 and 2). This is because Ces1c, a carboxylase that recognizes the VC structure and cleaves the amide bond at the carboxy terminal side of citrulline, exists in mouse plasma, so the linker containing the above dipeptide is cleaved in the plasma of Ces1c. Because it is thrown away. Therefore, for ADCs having linkers containing the above dipeptides, the in vivo dynamics are greatly different between mice and humans. Therefore, there is a problem that it is difficult to evaluate drug efficacy in humans in mice.

As described above, in order to improve the instability in mouse plasma of the ADC having the structure "antibody-spacer-VC structure-spacer-drug", the ADC was modified by modifying the linker (i.e., spacer-VC structure-spacer). Stabilization is being attempted.

For example, Patent Document 5 and Non-Patent Document 3 disclose stabilizing the linker by introducing a hydrophilic group (PEG) into the spacer between the VC structure and the drug (near the cleavage site by Ces1c).

Meanwhile, a technology for stabilizing the linker by introducing a specific group into the spacer existing between the antibody and the VC structure has also been reported. An example of a linker stabilized by this technique is one in which at least one α-amino acid residue (i.e., It is a linker of the following 1) to 4) characterized by including a structure (i.e., a structure represented by X-Val-Cit or NH-C(R)-CO-Val-Cit) linked by:

1) A linker containing a tripeptide structure (Glu-Val-Cit) in which a glutamic acid residue is linked to the N terminus of Val (patent document 6);

2) a linker containing a structure (Asp-Val-Cit) in which an aspartic acid residue is connected to the N terminus of Val (Non-patent Document 4);

3) a linker containing a structure represented by NH-C(R)-CO-Val-Cit (R represents a side chain having a hydrophilic group such as PEG) (Patent Documents 7 to 9);

4) a linker containing a structure represented by NH-C(R)-CO-Val-Cit (R represents a side chain having a hydrophilic group such as a sulfonic acid group or sugar) (patent document 10); and

5) A linker containing a structure represented by NH-C(R)-CO-Val-Cit (R represents a side chain having a cyclodextrin) (Patent Document 11).

In addition, as another example of a linker in which a specific group is introduced into the spacer existing between the antibody and the VC structure, the linkers 6) to 8) below have been reported:

6) C(M)-CO-Val-Cit (where, C in C(M) represents a carbon atom, M in C(M) represents a stability regulator containing a side chain such as an aromatic ring group, and CO represents a carbonyl group that is connected to the amino group of the valine residue to form an amide bond. A linker containing a structure represented by (Patent Document 12);

7) A special, highly controlled PEG linker containing PEG in both the main chain and the side chain of the spacer present between the antibody and the VC structure (Non-Patent Document 5), and

8) C(R _i )-NH-CO-, in which the structure called C(R i)-NH is linked to the position on the antibody side of the VC analog structure (CO-R _{ii -CO} -Cit) by an amide bond. A linker comprising a structure represented by R _ii -CO-Cit (where R _i represents a thiophenyl group and R _ii represents a cyclobutyl ring) (Patent Document 13).

However, Non-Patent Document 6 describes that the higher the hydrophobicity of ADC, the faster the plasma clearance, and that the hydrophobicity of ADC can be evaluated by HIC (Hydrophobic Interaction Chromatography)-HPLC.

International Publication No. 2018/199337 International Publication No. 2019/240288 International Publication No. 2019/240287 International Publication No. 2020/090979 US Patent Application Publication No. 2019/0365915 Specification International Publication No. 2018/218004 International Publication No. 2019/094395 US Patent Application Publication No. 2016/0310612 Specification International Publication No. 2020/252043 International Publication No. 2020/236841 International Publication No. 2018/213077 Japanese Patent Publication No. 2016-050204 International Publication No. 2016/090038

Dorywalska et al., Bioconjugate Chem., 2015, 26(4), 650-659 Dorywalska et al., Mol Cancer Ther., 2016, 15(5), 958-70 Poudel et al., ACS Med Chem Lett., 2020, 11(11), 2190-2194 Ratnayake et al., Bioconjug Chem., 2019, 30(1), 200-209 Walker et al., Bioconjug Chem. 2019, 30(11), 2982-2988 Lyon et al., Nat Biotechnol., 2015, 33(7), 733-5

The purpose of the present invention is to provide a conjugate of an antibody and a functional material or a salt thereof having excellent desired properties while controlling the binding ratio between the antibody and the functional material within a desired range.

As a result of careful study, the present inventors selected a lysine residue in the heavy chain of an immunoglobulin unit as the modification site for the antibody, and also selected a specific structural unit [i.e., CO-(NR ₁ )nX-CONH-CH ₂ (described later) By using a linker containing [R _A )-CONH-CH ₂ (-R _B )-CO] to connect the immunoglobulin unit and the functional substance, the average ratio of the combination of the immunoglobulin unit and the functional substance (functional substance/immune It was discovered that a conjugate represented by the formula (I) or a salt thereof having excellent desired properties can be easily produced while highly controlling the globulin unit) to the desired range (1.5 to 2.5). For example, such conjugates or salts thereof include one or more conjugates selected from the group consisting of excellent clearance (i.e., long residence time in the body), low aggregation rate, high cleavage by cathepsin B, and high stability in mouse plasma. It can have the desired properties.

The present inventors also discovered that, among the conjugates represented by the formula (I), the conjugates represented by the formulas (I-1) to (I-3) can be excellent in the desired properties, and completed the present invention. It came down to this.

For example, the conjugates represented by formulas (I-1) and (I-2) have a structural unit in which a tertiary amide-type structure with a group containing a hydrophilic group is linked to the VC structure, and the antibody and the functional material are linked. Among the linkers for this purpose, the prior art does not teach or suggest the conjugate of the present invention containing a linker having such a structural unit.

In addition, the conjugate represented by the formula (I-3) has a structural unit in which a γ glutamic acid residue is linked to the VC structure in the linker for connecting the antibody and the functional material, and the prior art has this structural unit. Conjugates containing linkers are neither taught nor suggested. In fact, Patent Documents 6 to 11 disclose a linker comprising a structural unit in which at least one α-amino acid residue (i.e., α-type structure) is connected to the VC structure, but a structural unit in which a γ glutamic acid residue is connected to the VC structure. The linker included is neither taught nor suggested. Patent Documents 12, 13, and Non-Patent Document 5 also do not teach or suggest a linker containing a structural unit in which a γ glutamic acid residue is connected to a VC structure.

That is, the present invention is as follows.

[1] A conjugate of an antibody and a functional substance, or a salt thereof, comprising a structural unit represented by the formula (I) and at least one hydrophilic group present in the structural unit.

[2] A conjugate of [1] or a salt thereof, wherein the immunoglobulin unit is a human immunoglobulin unit.

[3] A conjugate of [2] or a salt thereof, wherein the human immunoglobulin unit is a human IgG antibody.

[4] A conjugate or a salt thereof of any one of [1] to [3], in which a lysine residue is present at positions 246/248, 288/290, or 317 according to Eu numbering.

[5] A conjugate or a salt thereof of any one of [1] to [4], where r is 1.9 to 2.1.

[6] At least one group selected from the group consisting of hydrophilic group, carboxylic acid group, sulfonic acid group, hydroxyl group, polyethylene glycol group, polysarcosine group and sugar moiety, any one of [1] to [5], or a conjugate thereof salt.

[7] A conjugate or a salt thereof of any one of [1] to [6], in which L ₁ represents a divalent group represented by formula (i).

[8] L ₃ and L ₄ are each independently -(C(R) ₂ ) _m -, a conjugate of [7] or a salt thereof.

[9] Y has the following structural formula:

[Here, white circles and black circles represent bonding hands,

If the bond in the white circle is bonded to L ₃ , the bond in the black circle is bonded to L ₄ ,

When the bond in the white circle is bonded to L ₄ , the bond in the black circle is bonded to L _3. Conjugate of [7] or [8], a divalent group represented by any of the following structural formulas: Gate or its salt.

[10] [1] to [1], where the structural unit represented by formula (I) is a structural unit represented by formula (I-1), (I-2), (I-3), or (I-4). 9] any one of the conjugates or salts thereof.

[11] A conjugate of [10] or a salt thereof, wherein the structural unit represented by formula (I) is a structural unit represented by formula (I-1), (I-2), or (I-3).

[12] The structural unit represented by formula (I-1), (I-2), (I-3) or (I-4) has formula (I-1a'), (I-1b'), (I Structures denoted as -1c'), (I-2'), (I-3a'), (I-3b'), (I-4'), (I-4b') or (I-4c') A conjugate of [10] or a salt thereof.

[13] Formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), (I-3b') or (I-4 The structural unit represented by ') has the formula (I-1a'-1), (I-1a'-2), (I-1b'-1), (I-1c'-1), (I-2 '-1), (I-2'-2), (I-3a'-1), (I-3a'-2), (I-3b'-1), (I-4a'-1), Conjugate or salt thereof of [12], which is a structural unit represented by (I-4b'-1), (I-4c'-1) or (I-4c'-2):

[14] L ₂ has the following structural formula:

(Here, black circles and white circles represent bonding hands,

The binding hand in the black circle is bonded to the carbonyl group adjacent to L ₂ ,

The binding hand in the white circle is connected to D,

E represents an electron-withdrawing group,

n2 is an integer of 1 to 4.) A divalent group expressed as a conjugate of any one of [1] to [13] or a salt thereof.

[15] A compound or a salt thereof represented by the formula (II-1), (II-2), (II-3), (II-4b') or (II-4c').

[16] The compound of [15] or a salt thereof, wherein the bioorthogonal functional group is a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue.

[17] Compounds represented by formula (II-1), (II-2) or (II-3) have formulas (II-1a'), (II-1b'), (II-1c'), (II) Compound of [15] or [16] or salt thereof, represented by -2'), (II-3a') or (II-3b'):

[18] Chemical formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a'), (II-3b'), (II-4b ') or (II-4c') has the formula (II-1a'-1), (II-1a'-2), (II-1b'-1), (II-1c'-1) , (II-2'-1), (II-2'-2), (II-3a'-1), (II-3a'-2), (II-3b'-1), (II-4b A compound or salt thereof of any one of [15] to [17], represented by '-1), (II-4c'-1) or (II-4c'-2):

[19] L ₂ has the following structural formula:

(Here, black circles and white circles represent bonding hands,

The binding hand in the black circle is bonded to the carbonyl group adjacent to L ₂ ,

The binding hand in the white circle is connected to D,

E represents an electron-withdrawing group,

n2 is an integer of 1 to 4.) A compound of any one of [15] to [18] or a salt thereof, which is a divalent group represented by:

[20] A reagent for derivatizing an antibody containing the compound of any one of [15] to [19] or a salt thereof.

The conjugate or salt thereof of the present invention can have excellent desired properties while highly controlling the average ratio of the bond between the immunoglobulin unit and the functional substance (functional substance/immunoglobulin unit) to a desired range (1.5 to 2.5). .

The compounds of the present invention or their salts and reagents are useful as synthetic intermediates in the production of the above conjugates.

1. Definition of general terms

In the present invention, the term “antibody” is as follows. Additionally, the term “immunoglobulin unit” corresponds to the divalent monomer unit that is the basic component of such antibodies, and is a unit containing two heavy chains and two light chains. Therefore, with respect to immunoglobulin units, their origin, type (polyclonal or monoclonal, isotype and full-length antibody or antibody fragment), antigen, position of lysine residues and definitions, examples and preferred examples of regioselectivity are given below. It is the same as that of the antibody described in .

The origin of the antibody is not particularly limited, and for example, it may be derived from an animal such as a mammal or bird (eg, chicken). Preferably, the immunoglobulin units are of mammalian origin. Such mammals include, for example, primates (e.g. humans, monkeys, chimpanzees), rodents (e.g. mice, rats, guinea pigs, hamsters, rabbits), pets (e.g. dogs, cats), and livestock (e.g. cattle). , pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, and more preferably humans.

The type of antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (eg, IgG, IgD, IgE) or a tetravalent or higher antibody (eg, IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Monoclonal antibodies include, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a given sugar chain has been added (e.g., antibodies modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence), and double antibodies. Specific antibodies, Fc region proteins, and Fc fusion proteins may be included. Isotypes of monoclonal antibodies include, for example, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, as a monoclonal antibody, a full-length antibody or an antibody fragment containing a variable region and a CH1 domain and a CH2 domain can be used, but a full-length antibody is preferred. The antibody is preferably a human IgG monoclonal antibody, and more preferably a human IgG full-length monoclonal antibody.

As the antigen of the antibody, any antigen can be used. For example, such antigens include proteins [oligopeptides and polypeptides. Proteins modified with biomolecules such as sugars (e.g., glycoproteins) may also be used], sugar chains, nucleic acids, and low-molecular-weight compounds. Preferably, the antibody may be an antibody whose antigen is a protein. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.

More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of disease target proteins include the following.

(1) Cancer area

PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folic acid Receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1/B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1 , GM2, glypican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA33 , Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1 ( CD157), P-cadherin, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2, CSF-1R , FGFR3, OX40, BCMA, ErbB3, CD137(4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2 -PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA/B, Thomsen-Friedenreich , CD39, CD37, CD73, CLEC12A, Lgr3, transferrin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor ProteiN, NaPi2b, Lewis blood group B antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 Integrin, TAG-72(CA72-4), CD70

(2) Autoimmune disease/inflammatory disease

IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP (Alpha4 Beta 7 Integrin), IL-23, GM-CSFR, TSLP , CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 Integrin, LIFHT

(3) Cranial nerve disease

CGRP, CD20, β-amyloid, β-amyloid protopibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC Virus

(4) Infectious diseases

Clostridium Difficile toxin B, cytomegalovirus, RS virus, LPS, S.Aureus Alpha-toxin, M2e protein, Psl, PcrV, S.Aureus toxin, influenza A, Alginate, Staphylococcus aureus, PD-L1, influenza B, acine Tobacter, F-protein, Env, CD3, pathogenic E. coli, Klebsiella, pneumococcus

(5) Hereditary/rare diseases

Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

(6) Eye disease

Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

(7) Bone and orthopedics area

Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

(8) Blood diseases

vWF, Factor IXa, Factor

(9) Other diseases

BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8 , IL-1β, AT2-R, IAPP

Specific examples of monoclonal antibodies include specific chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoximab), and specific humanized antibodies. Antibodies (e.g., daclizumab, palivizumab, trastuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), Zumab, Pertuzumab, Obinutuzumab, Vedolizumab, Pembrolizumab (IgG4), Mepolizumab, Elotuzumab, Daratumumab, Ixekizumab (IgG4), Reslizumab (IgG4), Atezolizumab ), certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, Raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), allylocumab, necitumumab, brodalumab (IgG2), and olalatumab). (If the IgG subtype is not mentioned, it is indicated as IgG1).

The position of amino acid residues in the antibody and the position of the heavy chain constant region (e.g. CH2 domain) follows EU numbering (see http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html). For example, when targeting human IgG, the lysine residue at position 246 corresponds to the 16th amino acid residue in the human IgG CH2 region, and the lysine residue at position 248 corresponds to the 18th amino acid residue in the human IgG CH2 region. Corresponds to , the lysine residue at position 288 corresponds to the 58th amino acid residue of the human IgG CH2 region, the lysine residue at position 290 corresponds to the 60th amino acid residue of the human IgG CH2 region, and the lysine at position 317 The residue corresponds to the 87th amino acid residue of the human IgG CH2 region. The designation at position 246/248 indicates that the lysine residue at position 246 or 248 is of interest. The designation at position 288/290 indicates that the lysine residue at position 288 or 290 is of interest.

According to the present invention, a specific lysine residue (e.g., a lysine residue at position 246/248, 288/290, or 317) in the heavy chain of an immunoglobulin unit constituting an antibody can be site-selectively modified (e.g., international See Publication No. 2018/199337, International Publication No. 2019/240288, International Publication No. 2019/240287, and International Publication No. 2020/090979). In this specification, “regioselectivity” or “regioselectivity” means that a given structural unit capable of binding to a specific amino acid residue in an antibody is specific in the antibody, even though the specific amino acid residue is not localized in a specific region. It refers to being omnipresent in an area. Therefore, expressions related to regioselectivity such as “having regioselectively,” “regioselectively binding,” and “binding with regioselectivity” refer to a given structural unit in the target region containing one or more specific amino acid residues. This means that the retention or binding rate is significantly higher than the retention or binding rate of the structural unit in a non-target region containing a plurality of amino acid residues homologous to the specific amino acid residue in the target region. This regioselectivity is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, It may be 97% or higher, 98% or higher, 99% or higher, 99.5% or higher, or 100%.

In the present invention, as long as a specific lysine residue in the heavy chain of the antibody is site-selectively modified, specific amino acid residues at other positions may be further site-selectively modified. For example, methods for site-selectively modifying specific amino acid residues at predetermined positions in antibodies include International Publication No. 2018/199337, International Publication No. 2019/240288, International Publication No. 2019/240287, and International Publication No. 2020. It is described in No. /090979. Such specific amino acid residues include amino acid residues (e.g., lysine residues, aspartic acid residues, glutamic acid residues, asparagine residues, glutamine residues, a threonine residue, a serine residue, a tyrosine residue, a cysteine residue), preferably a lysine residue having a side chain containing an amino group, a tyrosine residue having a side chain containing a hydroxy group, a serine residue and a threonine residue, or It is a cysteine residue having a side chain containing a thiol group, and more preferably, it may be a lysine residue (i.e., two lysine residues among the lysine residues at positions 246/248, the lysine residues at positions 288/290, and the lysine residues at positions 317. may be position-selectively double-modified, or the three lysine residues may be position-selectively triple-modified).

(halogen atom)

Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

(Gi 1)

Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.

The monovalent group is one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, especially preferably 1 to 3). It may be substituted by a substituent described later.

(Monovalent hydrocarbon group and related terms)

Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

A monovalent chain hydrocarbon group refers to a hydrocarbon group composed only of a chain structure and does not contain a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of monovalent chain hydrocarbon groups include alkyl, alkenyl, and alkynyl. Alkyl, alkenyl, and alkynyl may be either linear or branched.

As alkyl, alkyl with 1 to 12 carbon atoms is preferable, alkyl with 1 to 6 carbon atoms is more preferable, and alkyl with 1 to 4 carbon atoms is still more preferable. When alkyl has a substituent, the number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, Examples include decyl and dodecyl.

As alkenyl, alkenyl with 2 to 12 carbon atoms is preferable, alkenyl with 2 to 6 carbon atoms is more preferable, and alkenyl with 2 to 4 carbon atoms is still more preferable. When alkenyl has a substituent, the number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

As alkynyl, alkynyl with 2 to 12 carbon atoms is preferable, alkynyl with 2 to 6 carbon atoms is more preferable, and alkynyl with 2 to 4 carbon atoms is still more preferable. When alkynyl has a substituent, the number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

As the monovalent chain hydrocarbon group, alkyl is preferable.

A monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbons may be either monocyclic or polycyclic. However, it does not need to be composed only of alicyclic hydrocarbons, and may contain a chain structure as part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, and these may be monocyclic or polycyclic.

As cycloalkyl, cycloalkyl with 3 to 12 carbon atoms is preferable, cycloalkyl with 3 to 6 carbon atoms is more preferable, and cycloalkyl with 5 to 6 carbon atoms is still more preferable. When cycloalkyl has a substituent, the number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

As cycloalkenyl, cycloalkenyl with 3 to 12 carbon atoms is preferable, cycloalkenyl with 3 to 6 carbon atoms is more preferable, and cycloalkenyl with 5 to 6 carbon atoms is still more preferable. When cycloalkenyl has a substituent, the number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

Cycloalkynyl is preferably cycloalkynyl with 3 to 12 carbon atoms, more preferably cycloalkynyl with 3 to 6 carbon atoms, and even more preferably cycloalkynyl with 5 to 6 carbon atoms. When cycloalkynyl has a substituent, the number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferable.

A monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it does not need to be composed only of aromatic rings, and may contain a chain structure or an alicyclic hydrocarbon in part, and the aromatic ring may be either monocyclic or polycyclic. As the monovalent aromatic hydrocarbon group, aryl with 6 to 12 carbon atoms is preferable, aryl with 6 to 10 carbon atoms is more preferable, and aryl with 6 carbon atoms is still more preferable. When the monovalent aromatic hydrocarbon group has a substituent, the number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of aryl having 6 to 12 carbon atoms include phenyl and naphthyl.

As the monovalent aromatic hydrocarbon group, phenyl is preferable.

Among these, preferred monovalent hydrocarbon groups include alkyl, cycloalkyl, and aryl.

(Monovalent heterocyclic group and related terms)

A monovalent heterocyclic group refers to a group in which one hydrogen atom has been removed from the heterocycle of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocyclic group preferably include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and are composed of an oxygen atom, a sulfur atom, and a nitrogen atom. It is more preferable to include one or more types selected from the group.

As the monovalent aromatic heterocyclic group, an aromatic heterocyclic group with 1 to 15 carbon atoms is preferable, an aromatic heterocyclic group with 1 to 9 carbon atoms is more preferable, and an aromatic heterocyclic group with 1 to 6 carbon atoms is still more preferable. When the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, Oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, furinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl and phthalazinyl. .

As the monovalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group with 2 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group with 2 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group with 2 to 6 carbon atoms is even more preferable. do. When the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of monovalent non-aromatic heterocyclic groups include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, and tetrahydro. Thiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, Hydrooxazinyl, tetrahydroxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

Among these, the monovalent heterocyclic group is preferably a 5-membered or 6-membered heterocyclic group.

(2 groups)

The divalent group is a divalent straight-chain hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR ₇ -, -C(=O)-NR ₇ -, -NR ₇ -C(-O)-, -C(=S)-NR ₇ -, -NR ₇ -C(=S)-, -O-, -S-, -(OR ₈ ) _m One group selected from the group consisting of - and -(SR ₈ ) _m1 -, or two or more of them (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, Even more preferably, it is a group having a main chain structure containing 2 to 5 groups, particularly preferably 2 or 3 groups. R ₇ represents a hydrogen atom or a substituent described later. R ₈ represents a divalent straight-chain hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. m1 is an integer of 1 to 10, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, even more preferably an integer of 1 to 5, especially preferably an integer of 1 to 3. It is an integer.

The divalent linear hydrocarbon group is linear alkylene, linear alkenylene, or linear alkynylene.

Straight-chain alkylene is a straight-chain alkylene having 1 to 6 carbon atoms, and linear alkylene having 1 to 4 carbon atoms is preferable. Examples of straight chain alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene.

Straight-chain alkenylene is linear alkenylene having 2 to 6 carbon atoms, and linear alkenylene having 2 to 4 carbon atoms is preferable. Examples of linear alkenylene include ethylenenylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene.

Straight-chain alkynylene is linear alkynylene having 2 to 6 carbon atoms, and linear alkynylene having 2 to 4 carbon atoms is preferable. Examples of straight-chain alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene.

As the divalent linear hydrocarbon group, linear alkylene is preferable.

The divalent cyclic hydrocarbon group is arylene or a divalent non-aromatic cyclic hydrocarbon group.

As arylene, arylene with 6 to 14 carbon atoms is preferable, arylene with 6 to 10 carbon atoms is more preferable, and arylene with 6 carbon atoms is particularly preferable. Examples of arylene include phenylene, naphthylene, and anthracenylene.

The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, and a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms is preferred. More preferred, a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms is particularly preferred. Examples of the divalent non-aromatic cyclic hydrocarbon group include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene.

As the divalent cyclic hydrocarbon group, arylene is preferable.

The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. As a heteroatom constituting the heterocycle, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and is composed of an oxygen atom, a sulfur atom, and a nitrogen atom. It is more preferable to include one or more types selected from the group.

As the divalent aromatic heterocyclic group, a divalent aromatic heterocyclic group having 3 to 15 carbon atoms is preferable, a divalent aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a divalent aromatic heterocyclic group having 3 to 6 carbon atoms is more preferable. Particularly desirable. Examples of divalent aromatic heterocyclic groups include pyrroldiyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazoldiyl, imidazolediyl, and thiazolediyl. , isothiazoldiyl, oxazoldiyl, isoxazoldiyl, triazoldiyl, tetrazoldiyl, indolediyl, purindiyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quina. Jolindiyl and phthalazidiyl may be mentioned.

As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group with 3 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group with 3 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group with 3 to 6 carbon atoms is particularly preferable. do. Examples of divalent non-aromatic heterocyclic groups include pyrrole dione diyl, pyrroline dione diyl, oxilandiyl, aziridine diyl, azetidine diyl, oxetane diyl, thiethane diyl, pyrrolidine diyl, and dihydrofurandiyl. , tetrahydrofurandiyl, dioxolandiyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidindiyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiyl Opyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydroxazinediyl, tetrahydroxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

As the divalent heterocyclic group, a divalent aromatic heterocyclic group is preferable.

Preferably, the divalent group is alkylene, arylene, -C(=O)-, -NR ₇ -, -C(=O)-NR ₇ -, -NR ₇ -C(=O)-, - It is a divalent group having a main chain structure containing one group selected from the group consisting of O- and -(OR ₈ ) _m- , or

Alkylene, arylene, -C(=O)-, -NR ₇ -, -C(=O)-NR ₇ -, -NR ₇ -C(=O)-, -O- and -(OR ₈ ) It is a divalent group having a main chain structure containing two or more groups selected from the group consisting of _m1 -,

R ₇ is a hydrogen atom or alkyl,

R ₈ is alkylene or arylene,

m1 may be an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5).

Alkylene, arylene, and alkyl are the same as described above.

The main chain structure in the divalent group is one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, especially preferred. In other words, it may be substituted by 1 to 3 substituents described later.

(substituent)

Substituents include:

(i) halogen atom;

(ii) monovalent hydrocarbon group;

(iii) monovalent heterocyclic group;

(iv) aralkyl;

(v) R _a -O-, R _a -C(=O)-, R _a -OC(=O)- or R _a -C(=O)-O-(R _a is a hydrogen atom or a monovalent hydrocarbon represents a flag.) or

(vi) NR _b R _c -, NR _b R _c -C(=O)-, NR _b R _c -C(=O)-O- or R _b -C(=O)-NR _c -(R _b and R _c are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group.);

(vii) nitro group, sulfuric acid group, sulfonic acid group, cyano group and carboxyl group.

The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, and monovalent heterocyclic group in the above substituents are the same as those described above.

Aralkyl refers to arylalkyl. The definitions, examples and preferred examples of aryl and alkyl in arylalkyl are as described above. As aralkyl, aralkyl having 3 to 15 carbon atoms is preferable. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

Preferably, the substituent may be as follows:

(i) halogen atom;

(ii) alkyl, phenyl or naphthyl having 1 to 12 carbon atoms;

(iii) aralkyl having 3 to 15 carbon atoms;

(iv) 5- or 6-membered heterocycle;

(v) R _a -O-, R _a -C(=O)-, R _a -OC(=O)- or R _a -C(=O)-O-(R _a is a hydrogen atom or the number of carbon atoms is 1 represents alkyl from 12 to 12.);

(vi) NR _b R _c -, NR _b R _c -C(=O)-, NR _b R _c -C(=O)-O- or R _b -C(=O)-NR _c -(R _b and R _c are the same or different and represent a hydrogen atom or an alkyl having 1 to 12 carbon atoms.) or

(vii) The same groups as listed in (vii) above.

More preferably, the substituents may be as follows:

(i) halogen atom;

(ii) alkyl having 1 to 12 carbon atoms;

(iii) R _a -O-, R _a -C(=O)-, R _a -OC(=O)- or R _a -C(=O)-O-(R _a is a hydrogen atom or the number of carbon atoms is 1 represents alkyl from 12 to 12.);

(iv) NR _b R _c -, NR _b R _c -C(=O)-, NR _b R _c -C(=O)-O- or R _b -C(=O)-NR _c -(R _b and R _c are the same or different and represent a hydrogen atom or an alkyl having 1 to 12 carbon atoms.) or

(v) The same groups as listed in (vii) above.

Even more preferably, the substituents may be:

(i) halogen atom;

(ii) alkyl having 1 to 6 carbon atoms;

(iii) R _a -O-, R _a -C(=O)-, R _a -OC(=O)- or R _a -C(=O)-O-(R _a is a hydrogen atom or the number of carbon atoms is 1 represents alkyl of to 6.);

(iv) NR _b R _c -, NR _b R _c -C(=O)-, NR _b R _c -C(=O)-O- or R _b -C(=O)-NR _c -(R _b and R _c are the same or different and represent a hydrogen atom or an alkyl having 1 to 6 carbon atoms.) or

(v) The same groups as listed in (vii) above.

Particularly preferably, the substituents may be as follows:

(i) halogen atom;

(ii) alkyl having 1 to 4 carbon atoms;

(iii) R _a -O-, R _a -C(=O)-, R _a -OC(=O)- or R _a -C(=O)-O-(R _a is a hydrogen atom or the number of carbon atoms is 1 represents alkyl of to 4.);

(iv) NR _b R _c -, NR _b R _c -C(=O)-, NR _b R _c -C(=O)-O- or R _b -C(=O)-NR _c -(R _b and R _c are the same or different and represent a hydrogen atom or an alkyl having 1 to 4 carbon atoms.) or

(v) The same groups as listed in (vii) above.

(hydrophilic group)

A hydrophilic group is a group that can make the structural unit represented by the formula (I) or a sub-concept formula thereof more hydrophilic. By having a hydrophilic group at a predetermined site in the structural unit, the conjugate can be more stabilized in mouse plasma. Examples of such hydrophilic groups include carboxylic acid groups, sulfonic acid groups, hydroxyl groups, polyethylene glycol groups, polysarcosine groups, and sugar moieties. One or more (e.g., 1, 2, 3, 4, or 5) hydrophilic groups may be included in the conjugate.

The polyethylene glycol group is a divalent group represented by -(CH ₂ -CH ₂ -O-) _k1 -. When the conjugate has a polyethylene glycol group, the conjugate may have a monovalent group in which one bond of the polyethylene glycol group is bonded to a hydrogen atom or a monovalent group (eg, a monovalent hydrocarbon group). k1 may be, for example, an integer of 3 or more, preferably an integer of 4 or more, more preferably an integer of 5 or more, and even more preferably an integer of 6 or more. k1 may also be an integer of 20 or less, preferably an integer of 15 or less, more preferably an integer of 12 or less, and even more preferably an integer of 10 or less. More specifically, k1 may be an integer of 3 to 20, preferably an integer of 4 to 15, more preferably an integer of 5 to 12, and even more preferably an integer of 6 to 10.

Polysarcosine group is a divalent group represented by -(NCH ₃ -CH ₂ -CO-) _k2 -. Polysarcosine group can be used as a substitute for PEG. k2 may be, for example, an integer of 3 or more, preferably an integer of 4 or more, more preferably an integer of 5 or more, and even more preferably an integer of 6 or more. k2 may also be an integer of 20 or less, preferably an integer of 15 or less, more preferably an integer of 12 or less, and even more preferably an integer of 10 or less. More specifically, k2 may be an integer of 3 to 20, preferably an integer of 4 to 15, more preferably an integer of 5 to 12, and even more preferably an integer of 6 to 10.

The sugar moiety is a monosaccharide, oligosaccharide (e.g., disaccharide, trisaccharide, tetrasaccharide, pentasaccharide), or polysaccharide. The sugar moiety may include aldose or ketose or combinations thereof. The sugar moiety may be a monosaccharide such as ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, or fructose, or an amino sugar (e.g., glucosamine), or an oligosaccharide or polysaccharide containing these monosaccharides.

In certain embodiments, the sugar moiety may be a low molecular weight hydrophilic group. A low molecular weight hydrophilic group refers to a hydrophilic group with a molecular weight of 1,500 or less. The molecular weight of the low molecular weight hydrophilic group is preferably 1,200 or less, 1,000 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less. Low-molecular-weight hydrophilic groups include carboxylic acid groups, sulfonic acid groups, hydroxyl groups, and polyethylene glycol groups, polysarcosine groups, and sugar moieties (e.g., monosaccharides, oligosaccharides) that fill the above molecular weight.

(bio-orthogonal functional group)

Bioorthogonal functional groups do not react with biological components (e.g. amino acids, proteins, nucleic acids, lipids, sugars, phosphoric acids) or have a slow reaction rate with biological components, but are selective for components other than biological components. refers to a group that reacts with Bioorthogonal functional groups are well known in the art (e.g., Sharpless KBet al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C. R. et al., Science 291, 2357 (2001); Bertozzi C. R. et al., Nature Chemical Biology 1, 13 (2005)).

In the present invention, as a bioorthogonal functional group, a functional group bioorthogonal to a protein is used. This is because the thiol group-introduced antibody that must be derivatized with the reagent of the present invention is a protein. A bioorthogonal functional group for a protein is a group that does not react with the side chains of the 20 natural amino acid residues constituting the protein or reacts with the side chains at a slow rate, but reacts with the functional group of interest. The 20 natural amino acids that make up proteins are alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), and methionine (M). ), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine ( R), histidine (H) and lysine (L). Among these 20 natural amino acids, glycine has no side chain (i.e., is a hydrogen atom) and has a side chain of a hydrocarbon group (i.e., the side chain does not contain a heteroatom selected from the group consisting of a sulfur atom, a nitrogen atom, and an oxygen atom). Alanine, isoleucine, leucine, phenylalanine and valine are inactive for most reactions. Therefore, bioorthogonal functional groups for proteins include asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, It is a group that does not react with the side chains of histidine and lysine, or has a slow reaction rate, but reacts with the target functional group.

Such bioorthogonal functional groups include, for example, an azide residue, an aldehyde residue, a thiol residue, an alkene residue (in other words, it may have a vinylene (ethenylene) moiety, which is the minimum unit having a double bond between carbon atoms. The same applies hereinafter. ), alkyne residue (in other words, it is good as long as it has an ethynylene moiety, which is the minimum unit with a triple bond between carbon atoms. The same applies hereinafter), halogen residue, tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine. moieties, ketone moieties, boronic acid moieties, cyanobenzothiazole moieties, allyl moieties, phosphine moieties, maleimide moieties, disulfide moieties, thioester moieties, α-halocarbonyl moieties (e.g., fluorine atom at the α position, chlorine atom) a carbonyl residue having an atom, a bromine atom, or an iodine atom (the same applies hereinafter), an isonitrile residue, a sidnon residue, and a selenium residue.

More specifically, the bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of the following.

〔here,

R _1a , single or multiple R _1b and single or multiple R _1c are the same or different and are the above substituents or electron-withdrawing groups,

· is a joining hand.〕

Examples of electron-withdrawing groups include halogen atoms, alkyl substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl, and keto groups (e.g., acyl). ), alkyloxy groups, and halogen atoms, boronic acid residues, mesyl, tosyl, and triflate are preferred.

The bioorthogonal functional group may be protected. A bioorthogonal functional group that may be protected refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group. The unprotected bioorthogonal functional group corresponds to the above bioorthogonal functional group. A protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleavage of the protecting group. Cleavage of the protecting group can be performed by specific treatment under conditions (mild conditions) that do not cause protein denaturation or decomposition (e.g., amide bond cleavage). Such specific treatments include, for example, (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) physical and chemical treatment selected from the group consisting of light. Examples include treatment with stimulation or (c) neglect when a cleavable linker containing a self-degradable cleavable moiety is used. These protecting groups and their cleavage conditions are common knowledge in the field (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Jounal of American Chemical Society. 132, 1500 (2010).; Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, J. M., Journal of American Chemical Society. 132, 17928 (2010); Thompson , D. H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R. G., Journal of Controlled Release, 95, 291 (2004).

Examples of protected bioorthogonal functional groups include disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and bisinaldiol residues.

More specifically, the protected bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of the following.

[Here, the dashed line perpendicular to the bond indicates the cleavage site,

Single or plural R _2a are the same or different and are selected from the group consisting of a hydrogen atom or the above substituents,

· is a joining hand.〕

Preferably, the bioorthogonal functional group that may be protected is an unprotected bioorthogonal functional group.

(functional substance)

The functional substance is not particularly limited as long as it is a substance that imparts a certain function to the antibody. Examples include drugs, labeling substances, and stabilizers, but drugs or labeling substances are preferred. The functional material may be a single functional material or a material in which two or more types of functional materials are connected.

The drug may be a drug for any disease. Such diseases include, for example, cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune Disease/inflammatory disease (e.g. allergic disease, rheumatoid arthritis, systemic erythematosus), cranial nerve disease (e.g. cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious disease (e.g. bacterial infection, viral infection), Hereditary/rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., macular degeneration, diabetic retinopathy, retinitis pigmentosa), diseases in the bone and orthopedic fields (e.g., arthrosis deformity), blood Examples include diseases (e.g., leukemia, purpura) and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver disease, kidney disease, lung disease, circulatory system disease, and digestive system disease). The drug may be a drug for the prevention or treatment of a disease or a drug for alleviating side effects.

More specifically, the drug may be an anticancer agent. Examples of anticancer agents include chemotherapy agents, toxins, radioactive isotopes, and substances containing these. Chemotherapeutic agents include, for example, DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalators, DNA cutting agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, and cytotoxic nucleosomes. Examples include seeds and platinum compounds. Examples of toxins include bacterial toxins (eg, diphtheria toxin) and plant toxins (eg, lysine). Radioactive isotopes include, for example, a radioisotope of a hydrogen atom (e.g. ³ H), a radioisotope of a carbon atom (e.g. ¹⁴ C), a radioisotope of a phosphorus atom (e.g. ³² P), and a radioisotope of a sulfur atom (e.g. , 35 ^S ), radioisotopes of yttrium (e.g. ⁹⁰ Y), radioisotopes of technetium (e.g. ^99m Tc), radioisotopes of indium (e.g. ¹¹¹ In), radioisotopes of the iodine atom (e.g. ¹²³ I, ¹²⁵ I, ¹²⁹ I, ¹³¹ I), radioisotope of samarium (e.g. ¹⁵³ Sm), radioisotope of rhenium (e.g. ¹⁸⁶ Re), radioisotope of astatine (e.g. ²¹¹ At), radioisotope of bismuth (e.g. ²¹² Bi) can be mentioned. More specifically, the drugs include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin flexible body, calicheamycin, duocamycin, eribulin, and anthrax. Cyclin, dmDNA31, and tubulysin can be mentioned.

A labeling substance is a substance that enables detection of a target (eg, tissue, cell, or substance). Labeling substances include, for example, enzymes (e.g. peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g. streptavidin, biotin, digoxigenin, aptamer), and fluorescence. Substances (e.g. fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g. luciferin, aequorin, acridinium ester, Tris(2,2'- Bipyridyl)ruthenium, luminol), radioactive isotopes (e.g., those described above), or substances containing them.

A stabilizer is a substance that enables stabilization of antibodies. Examples of stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

The functional substance may also be a peptide, protein, nucleic acid, low molecular weight organic compound, sugar chain, lipid, high molecular weight polymer, metal (eg, gold), or chelator. Examples of peptides include cell membrane-penetrating peptides, blood-brain barrier-penetrating peptides, and peptide pharmaceuticals. Examples of proteins include enzymes, cytokines, fragmented antibodies, lectins, interferons, serum albumin, and antibodies. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Nucleic acids also include, for example, RNA interference-inducing nucleic acids (eg, siRNA), aptamers, and antisense. Examples of low-molecular-weight organic compounds include protein degradation-inducing chimeric molecules, pigments, and photodegradable compounds.

(salt)

In the present invention, the term "salt" includes, for example, a salt with an inorganic acid, a salt with an organic acid, a salt with an inorganic base, a salt with an organic base, and a salt with an amino acid. Examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. You can. Examples of salts with inorganic bases include alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and salts with other metals such as zinc and aluminum, and ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. You can. Examples of salts with amino acids include salts with basic amino acids (eg, arginine, histidine, lysine, ornithine) and acidic amino acids (eg, aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (eg, hydrogen chloride) or a salt with an organic acid (eg, trifluoroacetic acid).

2. Conjugate or salt thereof

The present invention provides a conjugate of an antibody and a functional substance or a salt thereof, which includes a structural unit represented by the following formula (I), and at least one hydrophilic group is present in the structural unit.

[Formula (I)]

〔During the meal,

Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and also forms an amide bond with a carbonyl group adjacent to Ig positionally selectively through the amino group in the side chain of the lysine residue in the two heavy chains,

L ₁ and L ₂ each represent a divalent group,

R ₁ represents a monovalent group that may contain a hydrophilic group,

X shows a good two -sized group even if you have a substituent, wherein the secondary group is 1 to 3, which constitutes the main chain portion connecting two atoms on both sides of X, and the substituent contains hydrophilic groups. It is a good one to have,

D represents a functional material,

R _A represents the side chain of the valine residue,

R _B represents the side chain of a citrulline residue or an alanine residue,

n is 0 or 1, and here, when n is ₁ , the substituent on

The average ratio r of the amide linkages per two heavy chains is 1.5 to 2.5.]

In Formula (I) and other chemical formulas presented in relation to the present invention, - (hyphen) indicates that two units (eg, atoms, groups) present on both sides are covalently bonded.

Antibodies contain the above immunoglobulin units. Such antibodies include, for example, IgG antibodies, IgD antibodies, and IgE antibodies, which contain two heavy chains and two light chains and immunoglobulin units with disulfide bonds between the heavy chains and between the heavy and light chains, and four heavy chains. and an IgA antibody comprising an immunoglobulin unit comprising four light chains, and also having disulfide bonds between the heavy chains and between the heavy and light chains, comprising eight heavy chains and eight light chains, and also between the heavy chains and between the heavy and light chains. Examples include IgM antibodies containing immunoglobulin units with disulfide bonds, but IgG antibodies (e.g., IgG1, IgG2, IgG3, IgG4) are preferred. The antibody is preferably a human IgG monoclonal antibody, and more preferably a human IgG full-length monoclonal antibody.

The divalent groups represented by L ₁ and L ₂ are the same as those described above.

The hydrophilic group and monovalent group in R ₁ are the same as those described above.

The divalent group in This main chain portion is composed of a structure containing a chain structure, a cyclic structure, or a combination thereof. When the main chain portion is a chain structure that does not contain a cyclic structure, the number of carbon atoms in the main chain portion can be determined by counting the number of carbon atoms in the chain structure. On the other hand, when the main chain part has a structure containing a cyclic structure, the predetermined number of carbon atoms constituting the cyclic structure can be determined by counting the number of carbon atoms in the main chain part. Specifically, the number of carbon atoms in the main chain portion of the cyclic structure can be determined by counting the number of carbon atoms in the shortest path connecting the two bond hands in the cyclic structure (for example, the bold numbers in (a) to (d) below). (see text path). When the main chain is a structure containing a combination of a chain structure and a cyclic structure, the number of atoms in the main chain is the number of atoms in the chain structure that does not contain a cyclic structure, and the number of atoms in the shortest path connecting two bond hands in the cyclic structure. It can be determined by summing with .

· is a joining hand.

In the case of (a), the shortest path is the path in bold, so the number of atoms in the divalent cyclic structure that can be counted as the number of carbon atoms in the main chain portion is 2.

In the case of (b), the shortest path is the path in bold, so the number of atoms in the divalent cyclic structure that can be counted as the number of carbon atoms in the main chain portion is 3.

In the case of (c), since both paths are the shortest paths (equal distances), the number of carbon atoms in the divalent cyclic structure that can be counted as the number of carbon atoms in the main chain portion is 4 (therefore, in the present invention, this structure is 2 in excluded from this group).

In the case of (d), since the path to the condensation site is the shortest path, the number of carbon atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 4 (therefore, in the present invention, this structure is excluded from).

The divalent group in

Preferably, the divalent group in

(1) a divalent straight-chain hydrocarbon group having 1 to 3 carbon atoms;

(2) divalent cyclic hydrocarbon group and

(3) A divalent group in which a divalent cyclic hydrocarbon group and a divalent straight-chain hydrocarbon group (one or two) having 1 or 2 carbon atoms are linked.

The divalent straight-chain hydrocarbon group having 1 to 3 carbon atoms includes straight-chain alkylene having 1 to 3 carbon atoms (e.g., methylene, ethylene, n-propylene), and straight-chain alkenylene having 2 or 3 carbon atoms (e.g., ethenylene, propenyl). lene) or a straight-chain alkynylene with 2 or 3 carbon atoms (e.g., ethynylene, n-propynylene). As the divalent straight-chain hydrocarbon group having 1 to 4 carbon atoms, linear alkylene having 1 to 3 carbon atoms is preferable.

The divalent cyclic hydrocarbon group is arylene or a divalent non-aromatic cyclic hydrocarbon group. By appropriately setting the two bonding hands in this divalent cyclic hydrocarbon group, the number of atoms constituting the main chain as described above can be set to be 3 to 5.

As arylene, arylene with 6 to 10 carbon atoms is preferable, and arylene with 6 carbon atoms is more preferable. Examples of arylene include phenylene and naphthylene.

The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, and a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms is preferred. More preferred, a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms is particularly preferred. Examples of the divalent non-aromatic cyclic hydrocarbon group include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene.

As the divalent cyclic hydrocarbon group, arylene is preferable.

The divalent straight-chain hydrocarbon group having 1 or 2 carbon atoms is a straight-chain alkylene having 1 or 2 carbon atoms (e.g., methylene, ethylene), a straight-chain alkenylene having 2 carbon atoms (ethenylene), or a straight-chain alkynylene having 2 carbon atoms (ethylene). Nilen). As the divalent straight-chain hydrocarbon group having 1 to 4 carbon atoms, linear alkylene having 1 to 4 carbon atoms is preferable.

Among the above (1) to (3), the divalent group in X is preferably (1) or (2), and (1) is more preferable.

Regarding the substituents that the divalent group in X may have, the hydrophilic group and monovalent group are the same as those described above.

The functional substance represented by D is the same as described above.

R _A represents the side chain of a valine residue (i.e. -CH(CH ₃ ) ₂ ).

R _B represents the side chain of a citrulline residue (i.e. -CH ₂ CH ₂ CH ₂ NHCONH ₂ ) or the side chain of an alanine residue (i.e. -CH ₃ ).

n is 0 or 1. When n is 0, the unit of NR ₁ does not exist, and X is directly bonded to two carbonyl groups. When n is ₁ , the substituent on This ring contains a nitrogen atom linked to R ₁ as a ring constituent atom. As such a ring, a 5-membered ring or a 6-membered ring is preferable. This ring may also be a non-aromatic ring or an aromatic ring, and a non-aromatic ring (eg, pyrrolidine, piperazine) is preferable.

r represents the average ratio of the amide linkages per two heavy chains and is 1.5 to 2.5. This average ratio is preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. This average ratio is also preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, this average ratio is preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1.

Among the structural units represented by formula (I), at least one (e.g., one, two, or three) hydrophilic group exists. Therefore, the “monovalent group that may contain a hydrophilic group” is the “monovalent group that may contain a hydrophilic group” represented by R ₁ and the “substituent” in the “divalent group that may optionally have a substituent” represented by "and at least one of _the "rings that may contain a hydrophilic group" when the substituent on

In a specific embodiment, L ₁ may represent a divalent group represented by the following formula (i).

-L ₄ -Y L ₃ - (i)

〔During the meal,

L ₃ and L ₄ are each independently, -(C(R) ₂ ) _m -, -(OC(R) ₂ -C(R) ₂ )m- and -(C(R) ₂ -C(R) ₂ -O) _m - and a divalent group selected from the group consisting of combinations thereof,

R is each independently a hydrogen atom, alkyl with 1 to 6 carbon atoms, alkenyl with 2 to 6 carbon atoms, or alkynyl with 2 to 6 carbon atoms,

m is an integer from 0 to 20,

Y is a divalent group produced by the reaction of two bioorthogonal functional groups that can react with each other.]

The alkyl with 1 to 6 carbon atoms, alkenyl with 2 to 6 carbon atoms, or alkynyl with 2 to 6 carbon atoms represented by R are the same as those described above.

m is an integer from 0 to 20. m is preferably an integer of 1 or more, more preferably an integer of 2 or more, an integer of 3 or more, an integer of 4 or more, or an integer of 5 or more. m is also preferably an integer of 15 or less, more preferably an integer of 12 or less, an integer of 10 or less, or an integer of 9 or less. Additionally, m can be set to a different integer for the divalent groups represented by L ₃ and L ₄ .

In a specific embodiment, L ₃ and L ₄ may each independently contain a divalent group represented by -(C(R) ₂ ) _m -.

Y is a divalent group produced by the reaction of two bioorthogonal functional groups that can react with each other. Since the combination of two bioorthogonal functional groups capable of reacting with each other is well known, a person skilled in the art can appropriately select such a combination and appropriately set the divalent group generated by the reaction of two bioorthogonal functional groups capable of reacting with each other. Combinations of bioorthogonal functional groups that can react with each other include, for example, a combination of a thiol residue and a maleimide residue, a combination of a furan residue and a maleimide residue, and a combination of a thiol residue and a halocarbonyl residue (halogen is converted to thiol by a substitution reaction). substituted), a combination of an alkyne residue (preferably a group having a triple bond between carbon atoms, which may be substituted by a substituent as described above) and an azide residue, a combination of a tetrazine residue and an alkene residue, a tetrazine residue and a combination of an alkyne residue, and a combination of a thiol residue and another thiol residue (disulfide bond). Therefore, Y is a group produced by the reaction of a thiol moiety with a maleimide moiety, a group produced by the reaction of a furan moiety with a maleimide moiety, a group produced by the reaction of a thiol moiety with a halocarbonyl moiety, an alkyne moiety, and A group produced by the reaction of an azide residue, a group produced by the reaction of a tetrazine residue and an alkene residue, or a disulfide group produced by a combination of a thiol residue and another thiol residue may be used.

In a specific embodiment, Y may be a divalent group represented by any of the following structural formulas.

[Here, white circles and black circles represent bonding hands,

If the bond in the white circle is bonded to L ₃ , the bond in the black circle is bonded to L ₄ ,

When the bond in the white circle is bonded to L ₄ , the bond in the black circle is bonded to L _3. 〕

In a preferred embodiment, the structural unit represented by the above formula (I) may be a structural unit represented by the following formula (I-1), (I-2), (I-3), or (I-4). This structural unit may have excellent performance as a conjugate among the structural units represented by the above formula (I) (see Examples).

[Formula (I-1)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

R ₁ represents a monovalent group containing a hydrophilic group.]

[Formula (I-2)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

R ₂ represents a monovalent group containing a hydrophilic group.]

[Formula (I-3)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

R ₃ represents a hydrogen atom, an alkyl having 1 to 6 carbon atoms, or a monovalent group containing a hydrophilic group,

R ₄ represents a monovalent group containing a hydrophilic group.]

[Formula (I-4)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

R ₁ represents a hydrogen atom or alkyl having 1 to 6 carbon atoms,

R ₆ represents a monovalent group containing a hydrophilic group.]

In the above formula (I-1), (I-2), (I-3) or (I-4), the monovalent group containing a hydrophilic group and the alkyl having 1 to 6 carbon atoms are the same as those described above.

In a preferred embodiment, the structural unit represented by the formula (I) may be a structural unit represented by the formula (I-1), (I-2), or (I-3). This structural unit may have excellent performance as a conjugate among the structural units represented by the above formula (I) (see Examples).

In one embodiment, in the above formula (I-3), R ₃ may represent a hydrogen atom or an alkyl having 1 to 6 carbon atoms, and R ₄ may represent a monovalent group containing a hydrophilic group.

In another embodiment, in the above formula (I-3), R ₃ and R ₄ may each independently represent a monovalent group containing a hydrophilic group.

In a more preferred embodiment, the structural unit represented by the formula (I-1), (I-2), (I-3) or (I-4) has the formula (I-1a'), (I-1b) '), (I-1c'), (I-2'), (I-3a'), (I-3b'), (I-4a'), (I-4b') or (I-4c' ) may also be a structural unit. This structural unit may have excellent performance as a conjugate among the structural units represented by the above formula (I) (see Examples).

[Formula (I-1a')]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group.];

[Formula (I-1b')]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

Two L ₅ each independently represent a bond or a divalent group,

Two HGs each independently represent a hydrophilic group.];

[Formula (I-1c')]

〔During the meal,

Ig, L ₂ , D, R _A , R _B , r are the same as in formula (I),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group,

L _1a and L _1b each independently represent a bond or a divalent group,

HG' represents a divalent hydrophilic group.];

[Formula (I-2')]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group.];

[Formula (I-3a')]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group.];

[Formula (I-3b')]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

HG represents a hydrophilic group.];

[Formula (I-4a')]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group.];

[Formula (I-4b')]

〔During the meal,

Ig, L ₂ , D, R _A , R _B , r are the same as in formula (I),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group,

L _1a and L _1b each independently represent a bond or a divalent group,

HG' represents a divalent hydrophilic group.];

[Formula (I-4c')]

〔During the meal,

Ig, L ₁ , D, R _A , R _B , r are the same as in formula (I),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group,

L ₂ ' represents a bond or divalent group,

E represents an electron-withdrawing group,

n2 is an integer from 1 to 4.].

In the formulas (I-1a') to (I-4c'), the definitions, examples, and preferred examples of the divalent group and the hydrophilic group are the same as those described above. Examples of the divalent hydrophilic group include polyethylene glycol group, polysarcosine group, and sugar moiety as described in the hydrophilic group above, with polyethylene glycol group and polysarcosine group being preferred, and polyethylene glycol group being more preferred. The electron-withdrawing group is the same as described above. The bonding position of the electron-withdrawing group to the phenyl ring is ortho-position, meta-position, or para-position with respect to the adjacent amino group (NH), the ortho-position or para-position is preferable, and the ortho-position is preferable. n2 is preferably 1 or 2. The definitions, examples, and preferred examples of the divalent hydrophilic group, electron-withdrawing group, bonding position thereof, and n2 are the same for other formulas.

In a preferred embodiment, the structural unit represented by “-L ₅ -HG” may be selected from the group consisting of the following.

(a) -OH

(b) -NH(CH ₂ CH ₂ O) ₈ CH ₃

(c) -NHCH ₂ CH ₂ COOH

In a specific embodiment, the structural unit represented by the formula (I-1a') may be the following structural unit.

[Formula (I-1a'-1)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

[Formula (I-1a'-2)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

In another specific embodiment, the structural unit represented by the formula (I-1b') may be the following structural unit.

[Formula (I-1b'-1)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

[Formula (I-1c'-1)]

〔During the meal,

Ig, L ₂ , D, R _A , R _B , r are the same as in formula (I),

L _1a and L _1b each independently represent a bond or a divalent group,

n1 is an integer from 3 to 20.]

In the formula (I-1c'-1), the definitions, examples and preferred examples of the divalent group are the same as those described above. n1 may be an integer of 3 or more, preferably an integer of 4 or more, more preferably an integer of 5 or more, and even more preferably an integer of 6 or more. n1 may also be an integer of 20 or less, preferably an integer of 15 or less, more preferably an integer of 12 or less, and even more preferably an integer of 10 or less. More specifically, n1 may be an integer of 3 to 20, preferably an integer of 4 to 15, more preferably an integer of 5 to 12, and even more preferably an integer of 6 to 10. The definition, examples, and preferred examples of n1 are the same for other formulas.

In another specific embodiment, the structural unit represented by the formula (I-2') may be the following structural unit.

[Formula (I-2'-1)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

[Formula (I-2'-2)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

In another specific embodiment, the structural unit represented by the formula (I-3a') may be the following structural unit.

[Formula (I-3a'-1)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

[Formula (I-3a'-2)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

In another specific embodiment, the structural unit represented by the formula (I-3b') may be the following structural unit.

[Formula (I-3b'-1)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

In another specific embodiment, the structural unit represented by the formula (I-4a') may be the following structural unit.

[Formula (I-4a'-1)]

〔During the meal,

Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I).]

In another specific embodiment, the structural unit represented by the formula (I-4b') may be the following structural unit.

[Formula (I-4b'-1)]

〔During the meal,

Ig, L ₂ , D, R _A , R _B , r are the same as in formula (I),

L _1a and L _1b each independently represent a bond or a divalent group,

n1 represents an integer from 3 to 20.]

In another specific embodiment, the structural unit represented by the formula (I-4c') may be the following structural unit.

[Formula (I-4c'-1)]

〔During the meal,

Ig, L ₁ , D, R _A , R _B , r are the same as in formula (I),

L ₂ ' represents a bond or a divalent group.] or

[Chemical formula (I-4c'-2)]

〔During the meal,

Ig, L ₁ , D, R _A , R _B , r are the same as in formula (I),

L ₂ ' represents a bond or a divalent group.]

In another preferred embodiment, in the above formula, L ₂ may be a divalent group represented by the following structural formula.

(Here, black circles and white circles represent bonding hands,

The binding hand in the black circle is bonded to the carbonyl group adjacent to L ₂ ,

The binding hand in the white circle is connected to D,

E represents an electron-withdrawing group,

n2 is an integer from 1 to 4.)

The definitions, examples, and preferred examples of the electron-withdrawing group, its bonding position, and n2 are the same as those described above.

In a more preferred embodiment, the structural unit represented by the formula (I) is the formula (I-1a'), (I-1b'), (I-2'), (I-3a'), (I -3b'), (I-4b') or (I-4c'), or a structural unit represented by a chemical formula corresponding to a sub-concept of these chemical formulas may be used. This structural unit may have excellent conjugate performance among the structural units represented by the above formula (I) (see Examples).

In a specific embodiment, the conjugate or salt thereof of the present invention has the desired property of being difficult to aggregate, and can be specified by the aggregation rate. More specifically, the aggregation rate of the conjugate of the present invention or its salt may be 5% or less. This is because according to the present invention, it is easy to avoid aggregation of antibodies. The aggregation rate is preferably 4.8% or less, more preferably 4.6% or less, even more preferably 4.4% or less, particularly preferably 4.2% or less, 4.0% or less, 3.8% or less, 3.6% or less, 3.4% or less. or less, 3.2% or less, 3.0% or less, 2.8% or less, 2.6% or less, 2.4% or less, 2.2% or less, or 2.0% or less. The aggregation rate of the antibody can be measured by size exclusion chromatography (SEC)-HPLC (see examples and ChemistrySelect, 2020, 5, 8435-8439).

The conjugate of the present invention or a salt thereof has the following formula (II):

[Formula (II)]

〔During the meal,

L ₂ and L ₃ each represent a divalent group,

R ₁ represents a monovalent group that may contain a hydrophilic group,

X shows a good two -stage group even if you have a substituent, wherein the secondary group has 1 to 3 carbon atoms that make up the main chain that connects two atoms on both sides of X, and the substituent contains hydrophilic groups. It is a good one to have,

B represents a bioorthogonal functional group,

D represents a functional material,

R _A represents the side chain of the valine residue,

R _B represents the side chain of a citrulline residue or an alanine residue,

n is 0 or 1, and here, when n is ₁ , the substituent on

It can be produced by reacting with a raw antibody containing an immunoglobulin unit containing two heavy chains and two light chains (here, the two heavy chains include a lysine residue regioselectively modified with a bioorthogonal functional group). Definitions, examples and _preferred examples for L ₂ , _{L 3} , R ₁ ,

The definition, examples, and preferred examples of the bioorthogonal functional group represented by B are as described above.

In a specific embodiment, the bioorthogonal functional group represented by B may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. The bioorthogonal functional group represented by B can be selected so that it can react with the bioorthogonal functional group (e.g., the bioorthogonal functional group represented by B') in the raw antibody described later.

In a preferred embodiment, the compound represented by the formula (II) or a salt thereof is a compound represented by the formula (II-1), (II-2), (II-3) or (II-4) or a salt thereof. It's okay too. These compounds or their salts are useful as synthetic intermediates in the production of conjugates that can exhibit excellent performance among the compounds or their salts represented by the above formula (II) (see Examples).

[Formula (II-1)]

〔During the meal,

L ₂ and L ₃ each represent a divalent group,

R ₁ represents a monovalent group that may contain a hydrophilic group,

D represents a functional material,

R _A represents the side chain of the valine residue,

R _B represents the side chain of a citrulline residue or an alanine residue,

B represents a bioorthogonal functional group.]

[Formula (II-2)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

R ₂ represents a monovalent group that may contain a hydrophilic group.]

[Formula (II-3)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

R ₃ represents a hydrogen atom, an alkyl having 1 to 6 carbon atoms, or a monovalent group containing a hydrophilic group,

R ₄ represents a monovalent group containing a hydrophilic group.]

[Formula (II-4)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

R ₁ represents a hydrogen atom or alkyl having 1 to 6 carbon atoms,

R ₆ represents a monovalent group containing a hydrophilic group.]

In the above formula (II-1), (II-2), (II-3) or (II-4), the monovalent group containing a hydrophilic group and the alkyl having 1 to 6 carbon atoms are the same as those described above.

In one embodiment, in the above formula (II-3), R ₃ may represent a hydrogen atom or an alkyl having 1 to 6 carbon atoms, and R ₄ may represent a monovalent group containing a hydrophilic group.

In another embodiment, in the above formula (II-3), R ₃ and R ₄ may each independently represent a monovalent group containing a hydrophilic group.

In a preferred embodiment, the compound represented by the formula (II) or a salt thereof may be a compound represented by the formula (II-1), (II-2), or (II-3), or a salt thereof. These compounds or their salts are useful as synthetic intermediates in the production of conjugates that can exhibit excellent performance, among the compounds or their salts represented by the above formula (II) (see Examples).

In a more preferred embodiment, the compound represented by the formula (II-1), (II-2), (II-3) or (II-4) or a salt thereof has the formula (II-1a'), (II -1b'), (II-1c'), (II-2'), (II-3a'), (II-3b'), (II-4a'), (II-4b') or (II- The compound represented by 4c') or a salt thereof may be used. These compounds or their salts are useful as synthetic intermediates in the production of conjugates that can exhibit excellent performance, among the compounds or their salts represented by the above formula (II) (see Examples).

[Formula (II-1a')]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group.]

[Formula (II-1b')]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

Two L ₅ each independently represent a bond or a divalent group,

The two HGs each independently represent a hydrophilic group.]

[Formula (II-1c')]

〔During the meal,

L ₂ , D, R _A , R _B and B are the same as in formula (II-1),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group,

L _3a and L _3b each independently represent a bond or a divalent group,

HG' represents a divalent hydrophilic group.]

[Formula (II-2')]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group.]

[Formula (II-3a')]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group.]

[Formula (II-3b')]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

HG represents a hydrophilic group.]

[Formula (II-4a')]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group.]

[Formula (II-4b')]

〔During the meal,

L ₂ , D, R _A , R _B and B are the same as in formula (II-1),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group,

L _3a and L _3b each independently represent a bond or a divalent group,

HG' represents a divalent hydrophilic group.]

[Formula (II-4c')]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

L ₅ represents a bond or a divalent group,

HG represents a hydrophilic group,

L ₂ ' represents a bond or divalent group,

E represents an electron-withdrawing group,

n2 is an integer from 1 to 4.]

In the formulas (II-1a') to (II-4c'), the definitions, examples, and preferred examples of the structural units represented by the divalent group and the hydrophilic group are the same as those described above. The definitions, examples, and preferred examples of the divalent hydrophilic group, electron-withdrawing group, bonding position thereof, and n2 are the same as those described above.

In a preferred embodiment, the structural unit represented by “-L ₅ -HG” may be selected from the group consisting of the following.

(a) -OH

(b) -NH(CH ₂ CH ₂ O) ₈ CH ₃

(c) -NHCH ₂ CH ₂ COOH

In a specific embodiment, the compound represented by formula (II-1a') may be as follows.

[Formula (II-1a'-1)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

[Formula (II-1a'-2)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

In another specific embodiment, the compound represented by formula (II-1b') may be as follows.

[Formula (II-1b'-1)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

In another specific embodiment, the compound represented by the formula (II-1c') may be as follows.

[Formula (II-1c'-1)]

〔During the meal,

L ₂ , D, R _A , R _B and B are the same as in formula (II-1),

L _3a and L _3b each independently represent a bond or a divalent group,

n1 is an integer from 3 to 20.]

The definitions, examples, and preferred examples of the divalent groups represented by L _3a and L _3b and n1 are the same as those described above (the same applies hereinafter).

In another specific embodiment, the compound represented by the formula (II-2') may be as follows.

[Formula (II-2'-1)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

[Formula (II-2'-2)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

In another specific embodiment, the compound represented by the formula (II-3a') may be as follows.

[Formula (II-3a'-1)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

[Formula (II-3a'-2)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

In another specific embodiment, the compound represented by the formula (II-3b') may be as follows.

[Formula (II-3b'-1)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

In another specific embodiment, the compound represented by the formula (II-4a') may be as follows.

[Formula (II-4a'-1)]

〔During the meal,

L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1).]

In another specific embodiment, the compound represented by the formula (II-4b') may be as follows.

[Formula (II-4b'-1)]

〔During the meal,

L ₂ , D, R _A , R _B and B are the same as in formula (II-1),

L _3a and L _3b each independently represent a bond or a divalent group,

n1 is an integer from 3 to 20.]

In another specific embodiment, the compound represented by formula (II-4c') may be as follows.

[Formula (II-4c'-1)]

〔During the meal,

L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

L ₂ ' represents a bond or a divalent group.] or

[Formula (II-4c'-2)]

〔During the meal,

L ₃ , D, R _A , R _B and B are the same as in formula (II-1),

L ₂ ' represents a bond or a divalent group.]

In a preferred embodiment, in the above formula, L ₂ may be a divalent group represented by the following structural formula.

(Here, black circles and white circles represent bonding hands,

The binding hand in the black circle is bonded to the carbonyl group adjacent to L ₂ ,

The binding hand in the white circle is connected to D,

E represents an electron-withdrawing group,

n2 is an integer from 1 to 4.)

The definitions, examples, and preferred examples of the electron-withdrawing group, its bonding position, and n2 are the same as those described above.

In a more preferred embodiment, the compound represented by the formula (II) or a salt thereof has the formula (II-1a'), (II-1b'), (II-2'), (II-3a'), Structural units expressed by (II-3b'), (II-4b') or (II-4c') or chemical formulas corresponding to subconcepts of these chemical formulas may also be used. These compounds or their salts are useful as synthetic intermediates in the production of conjugates that can exhibit excellent performance, among the compounds or their salts represented by the above formula (II) (see Examples).

The raw antibody contains a lysine residue that is site-selectively modified with a bioorthogonal functional group. The bioorthogonal functional group in the raw antibody may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. The bioorthogonal functional group in the raw antibody can be selected so that it can react with the bioorthogonal functional group in the compound or salt thereof (e.g., the bioorthogonal functional group represented by B).

In certain embodiments, the source antibody has the formula (III):

[Formula (III)]

〔During the meal,

Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and also forms an amide bond with a carbonyl group adjacent to Ig positionally selectively through the amino group in the side chain of the lysine residue in the two heavy chains,

L ₄ is -(C(R) ₂ ) _m -, -(OC(R) ₂ -C(R) ₂ ) _m - and -(C(R) ₂ -C(R) ₂ -O) _m - 2 is a group selected from the group consisting of,

R is each independently a hydrogen atom, alkyl with 1 to 6 carbon atoms, alkenyl with 2 to 6 carbon atoms, or alkynyl with 2 to 6 carbon atoms,

m is an integer from 0 to 10,

B' is a bioorthogonal functional group capable of reacting with the bioorthogonal functional group represented by B,

The average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5.] may also contain an immunoglobulin unit represented by. The definitions, examples and preferred examples for Ig, L ₄ , R, m and r in the above formula (III) are the same as those described above.

The bioorthogonal functional group represented by B' is the same as the bioorthogonal functional group described above with respect to the raw antibody.

The above reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of proteins (e.g., cleavage of amide bonds). For example, this reaction can be carried out at room temperature (eg, about 15 to 30° C.) in an appropriate reaction system, for example, a buffer solution. The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, even more preferably 30 minutes to 8 hours. For details of this reaction, For example, G. J. L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G. J. L. Bernardes et al., Chem. Asian. J., 4, 630 (2009); B. G. Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. See Chem., 25, 825 (2014).

Confirmation of the production of the conjugate or its salt depends on the specific raw materials and molecular weight of the product, but can be performed, for example, by reverse-phase HPLC or mass spectrometry under reducing conditions. The conjugate or its salt can be appropriately purified by any method such as chromatography (eg, affinity chromatography).

The conjugate of the present invention or its salt can be used, for example, as a medicine or reagent (eg, diagnostic drug, research reagent).

The conjugate of the present invention or its salt may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the conjugate of the present invention or a salt thereof. Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, and polypropyl cellulose. Binders such as pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, disintegrants such as starch, carboxymethyl cellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, calcium citrate, magnesium stearate, Aero Lubricants such as vagina, talc, and sodium lauryl sulfate, fragrances such as citric acid, menthol, glycylysine/ammonium salt, glycine, and orange powder, preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben, citric acid, and sodium citrate. , stabilizers such as acetic acid, suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate, dispersants such as surfactants, diluents such as water, physiological saline, and orange juice, and bases such as cacao oil, polyethylene glycol, and white kerosene. Wax and the like can be mentioned, but are not limited to these. The conjugate or salt thereof of the present invention may also have any modification (eg, PEGylation) that achieves stability.

Formulations suitable for oral administration include solutions in which an effective amount of the ligand is dissolved in a diluent such as water, saline, or orange juice, capsules, sachets or tablets containing an effective amount of the ligand as a solid or granule, and an effective amount in a suitable dispersion medium. These include a suspension in which the active ingredient is suspended, and an emulsion in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a suitable dispersion medium.

The pharmaceutical composition is suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents, etc. Additionally, aqueous and non-aqueous sterile suspension preparations may be included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptance, body weight, age, etc. of the administration target, but can be set appropriately.

3. Compounds or salts thereof and reagents containing them

The present invention also provides a compound represented by the above formula (II-1), (II-2) or (II-3) or a salt thereof. The details of these compounds or their salts are the same as those described above.

In a preferred embodiment, the compound represented by the formula (II-1), (II-2), or (II-3) or a salt thereof is represented by the formula (II-1a'), (II-1b'), ( A compound represented by a chemical formula equivalent to II-2'), (II-3a') or (II-3b') or a sub-concept of these chemical formulas, or a salt thereof may be used. The details of these compounds or their salts are the same as those described above.

The compounds of the present invention or salts thereof are useful as synthetic intermediates in the preparation of conjugates that can exhibit excellent performance (see Examples).

The present invention also provides an antibody comprising a compound represented by the above formula (II-1), (II-2), (II-3), (II-4b') or (II-4c') or a salt thereof. A derivatization reagent is provided.

Details of the raw antibody or salt thereof derivatized with the reagent of the present invention and the conjugate or salt thereof obtained by the derivatization reaction of the raw antibody or salt thereof are as described above.

The reagent of the present invention may be provided in the form of a composition that additionally contains other components. Examples of such other components include solutions and stabilizers (eg, antioxidants and preservatives). As the solution, an aqueous solution is preferable. Aqueous solutions include, for example, water (e.g., distilled water, sterilized distilled water, purified water, physiological saline), buffer solutions (e.g., phosphoric acid aqueous solution, Tris-hydrochloric acid buffer, carbonate-bicarbonate buffer, boric acid aqueous solution, glycine-sodium hydroxide buffer, citric acid buffer). ), but a buffer solution is preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The reagent of the present invention can be provided in liquid or powder form (e.g., freeze-dried powder).

Example

The present invention will be explained in more detail by referring to examples below, but the present invention is not limited to the following examples.

Example 1: Synthesis of Linker-payload mimic

(1-1) Synthesis of Linker-payload mimic (1)

Linker-payload mimic (1) was synthesized as follows.

(1-1-1) Synthesis of pyrene (2)

Fmoc-Val-Cit-PAB-PNP (CAS No: 863971-53-2, 121.2 mg, 0.15 mmol) was dissolved in N,N-dimethylformamide (5 mL), and sarcosine-pyrene (as described in WO2018218004A1) was dissolved in 59.2mg, 0.196mmol), N,N-diisopropylethylamine (39μL, 0.227mmol), and 4-dimethylaminopyridine (3.7mg, 0.03mmol) were added, stirred at room temperature for 2 hours, and then diethylamine (2mL, 18.95mmol) was added and stirred at room temperature for 1.5 hours. After concentration under reduced pressure, it was purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above pyrene (2) (73.7 mg, 0.104 mmol).

¹ H NMR (400MHz, DMSO-d6) δ10.21(s, 1H), 8.68(s, 1H), 8.43-7.93(m, 12H), 7.60(t, J=7.1Hz, 2H), 7.31(m) ，2H)，6.04(s，1H)，5.48(s，2H)，5.02(d，J=16.1Hz，4H)，4.54(s，1H)，3.96(s，2H)，3.66(s，2H) ，2.92(d，J=6.1Hz，3H)，2.08(q，J=6.6Hz，1H)，1.80-1.56(m，2H)，1.46(s，2H)，1.21-1.13(m，1H)， 0.94(dt，J=6.8，3.0Hz，6H).

MS(ESI)m/z: 708.80[M+H] ⁺

(1-1-2) Synthesis of pyrene (3)

Fmoc-Glu(OtBu)-OH·H ₂ O (11.1 mg, 0.025 mmol) was dissolved in dimethylformamide (1 mL), pyrene (2) (17.3 mg, 0.024 mmol), 1-hydroxy-7-aza Benzotriazole (5.1mg, 0.037mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.3mg, 0.038mmol), and triethylamine (7.1μL, 0.51mmol) were added and cooled to room temperature. After stirring for 2.5 hours, diethylamine (0.2 mL, 1.91 mmol) was added and stirred at room temperature for 1.5 hours. After concentration under reduced pressure, it was purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain pyrene (3) (15.6 mg, 0.017 mmol).

¹ H NMR (400MHz, Chloroform-d) δ10.87(s, 1H), 8.68(m, 1H), 8.41-7.97(m, 13H), 7.60-7.58(m, 2H), 7.30(m, 2H) ，6.00(t，J=6.2Hz，1H)，5.45(s，2H)，5.05-4.99(m，4H)，4.46(m，1H)，4.26(m，1H)，3.96(s，2H)， 3.88(m，1H)，3.07(m，1H)，2.96(m，1H)，2.93(d，J=5.6Hz，3H)，2.68(t，J=1.8Hz，1H)，2.34-2.30(m ，3H)，2.03(m，1H)，1.93-1.90(m，2H)，1.30(s，9H)，1.15(s，1H)，0.92-0.86(m，6H).

MS(ESI)m/z: 893.45[M+H] ⁺

(1-1-3) Synthesis of pyrene (4)

Pyrene (3) (15.6 mg, 0.017 mmol) was dissolved in dimethylformamide (1.5 mL), 6-maleimide hexanoic acid (3.7 mg, 0.018 mmol), 1-hydroxy-7-azabenzotriazole (3.5 mg, 0.025mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.8mg, 0.025mmol), and triethylamine (4.8μL, 0.34mmol) were added and stirred at room temperature for 3 hours. Afterwards, 6-maleimide hexanoic acid (1.8mg, 0.009mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3mg, 0.012mmol), triethylamine (2.4μL, 0.17mmol) ) was added and stirred at room temperature for 1.5 hours. After that, 4-dimethylaminopyridine (0.5 mg, 0.004 mmol) was added and stirred for an additional 2.5 hours. After concentration under reduced pressure, it was purified by column chromatography (dichloromethane:methanol=9:1). The fraction containing the product was recovered and concentrated under reduced pressure to obtain the above pyrene (4) (8.0 mg, 0.007 mmol).

MS(ESI)m/z: 1086.60[M+H] ⁺

(1-1-4) Synthesis of Linker-payload mimic (1)

Pyrene (4) (9.7 mg, 0.0089 mmol) was dissolved in 1,4-dioxane (1 mL) and hydrogen chloride/1,4-dioxane solution (1 mL), and stirred in an ice water bath for 2 hours. Dimethylformamide (1 mL) was added, allowed to rise to room temperature, and then concentrated under vacuum. Afterwards, it was purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload mimic (1) (2.0 mg, 0.002 mmol).

MS(ESI)m/z: 1030.50[M+H] ⁺

(1-2) Synthesis of Linker-payload mimic (5)

Linker-payload mimic (5) was synthesized as follows.

(1-2-1) Synthesis of maleimide (6)

Fmoc-Glu-OtBu (213.0 mg, 0.50 mmol) was dissolved in dichloromethane (2 mL), N-(5-aminopentyl) maleimide hydrochloride (108.7 mg, 0.48 mmol), 1-hydroxybenzotriazole (101.7 mg, 0.753mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (143.6mg, 0.749mmol), triethylamine (155μL, 1.12mmol), 4-dimethylaminopyridine (7.2mg, 0.0589 mmol) was added and stirred at room temperature for 2 hours. After concentration under reduced pressure, it was purified by column chromatography (hexane:ethyl acetate=4:1). The fraction containing the product was recovered and concentrated under reduced pressure to obtain the maleimide (6) (254.1 mg, 0.431 mmol).

¹ H NMR (400MHz, Methanol-d4) δ7.82(d, J=7.6Hz, 3H), 7.70(t, J=6.7 Hz, 2H), 7.41(t, J=7.5Hz, 2H), 7.37- 7.29(m，2H)，6.79(s，2H)，4.43(dd，J=10.4，6.8Hz，1H)，4.34(dd，J=10.4，7.0Hz，1H)，4.24(t，J=6.9Hz ，1H)，4.18-4.00(m，1H)，3.50(t，J=7.1Hz，2H)，3.16(td，J=6.9，2.0Hz，2H)，2.28(t，J=7.5Hz，2H) ，2.24-2.08(m，1H)，1.99-1.83(m，1H)，1.48(s，13H)，1.28(dt，J=17.1，7.1Hz，2H).

MS(ESI)m/z: 590.25[M+H] ⁺

(1-2-2) Synthesis of maleimide (7)

Maleimide (6) (253.1 mg, 0.41 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, 1M hydrochloric acid (2 mL) was added, stirred at room temperature for 3 minutes, concentrated under reduced pressure, and freeze-dried to obtain the above-mentioned maleimide (7) (244.6 mg, 0.458 mmol).

¹ H NMR (400MHz, Methanol-d4) δ7.82 (d, J = 7.5Hz, 2H), 7.70 (dt, J = 9.3, 4.7Hz, 2H), 7.41 (t, J = 7.5Hz, 2H), 7.33(t，J=7.4Hz，2H)，6.79(s，2H)，4.49-4.30(m，2H)，4.30-4.11(m，2H)，3.50(t，J=7.0Hz，2H)，3.16 (t，J=7.2Hz，2H)，2.36-2.09(m，3H)，2.09-1.86(m，1H)，1.56(dt，J=25.5，7.5Hz，5H)，1.39-1.23(m，2H) ).

MS(ESI)m/z: 534.20[M+H] ⁺

(1-2-3) Synthesis of pyrene (8)

Maleimide (7) (31.5 mg, 0.052 mmol) was dissolved in N, N-dimethylformamide (2 mL), pyrene (2) (35.5 mg, 0.50 mmol) synthesized in Example 1-1-1, 1 -Hydroxybenzotriazole (10.1mg, 0.0747mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride (15.0mg, 0.0782mmol), triethylamine (14μL, 0.10mmol), 4 -Dimethylaminopyridine (0.7 mg, 0.00573 mmol) was added, stirred at room temperature for 2 hours, and purified by column chromatography (hexane:ethyl acetate=15:85). The fraction containing the product was recovered and concentrated under reduced pressure to obtain the above pyrene (8) (7.4 mg, 6.05 nmol).

MS(ESI)m/z: 1223.65[M+H] ⁺ ，612.65[M+2H] ²⁺

(1-2-4) Synthesis of Linker-payload mimic (5)

Pyrene (8) (6.3 mg, 0.0051 mmol) was dissolved in N,N-dimethylformamide (0.6 mL), dicyclohexylamine (0.12 mL, 0.6 mmol) was added, and stirred at room temperature for 3 hours. Anhydrous succinic acid (1.0 mg, 0.0095 mmol) was added, stirred at room temperature for 1.5 hours, and then purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload mimic (5) (1.10 mg, 0.991 nmol).

¹ H NMR (400MHz, DMSO-d6) δ10.01(s, 1H), 8.59(d, J=6.2Hz, 1H), 8.36-7.87(m, 10H), 7.67(t, J=8.7Hz, 2H )，7.22(dd，J=26.1，8.1Hz，3H)，6.91(s，2H)，5.92(s，1H)，4.94(d，J=18.4Hz，4H)，4.33(d，J=7.5Hz ，1H)，3.88(s，2H)，3.10(s，3H)，3.05-2.78(m，7H)，2.69(d，J=22.3Hz，1H)，2.38-2.22(m，3H)，2.15- 1.89(m，4H)，1.81(d，J=14.3Hz，1H)，1.72-1.50(m，3H)，1.48-1.22(m，6H)，1.14(dd，J=35.5，8.5Hz，4H) ，0.92(s，1H)，0.85-0.68(m，6H).

MS(ESI)m/z: 1101.55[M+H] ⁺

(1-3) Synthesis of Linker-payload mimic (9)

Linker-payload mimic (9) was synthesized as follows.

(1-3-1) Synthesis of maleimide (10)

6-maleimide hexanoic acid (300 mg, 1.420 mmol) was dissolved in dimethylformamide (2 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazoro[4,5-b ]Pyridinium 3-oxide hexafluorophosphate (647.8mg, 1.704mmol), imino diacetic acid (189.0mg, 1.420mmol), N,N-diisopropylethylamine (0.29mL, 1.704mmol), 4-dimethyl Aminopyridine (34.7 mg, 0.284 mmol) was added and stirred for 1 hour. After concentration under reduced pressure, it was purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain maleimide (10) (65.9 mg, 0.202 mmol).

¹ H NMR (400MHz, DMSO-d6) δ6.79(s, 2H), 4.25(s, 2H), 4.11(s, 2H), 3.49(t, J=7.0Hz, 2H), 2.35(t, J) =7.4Hz，2H)，1.16(m，4H)，1.33(m，2H)

MS(ESI)m/z: 327.00[M+H] ⁺

(1-3-2) Synthesis of Linker-payload mimic (9)

Maleimide (10) (46.2 mg, 0.142 mmol) was dissolved in dimethylformamide (0.3 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazoro[4,5- b] Pyridinium 3-oxide hexafluorophosphate (54.0 mg, 0.142 mmol) was added and stirred for 1 hour, then pyrene (2) (50.1 mg, 0.71 mmol) synthesized in Example 1-1-1, A dimethylformamide solution (0.3 mL) of N,N-diisopropylethylamine (0.025 mL, 0.142 mmol) was added, and the mixture was stirred for an additional 3 hours. After purification by reverse-phase preparative chromatography, the fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload mimic (9) (24.4 mg, 0.024 mmol). .

MS(ESI)m/z: 1016.50[M+H] ⁺

Example 2: Synthesis of Linker-Payload

(2-1) Synthesis of Linker-payload(11)

Linker-payload (11) was synthesized using the known material Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) according to Example 1-1.

MS(ESI)m/z: 1445.8[M+H] ⁺

(2-2) Synthesis of Linker-payload(12)

Linker-payload (12) was synthesized as follows.

Fmoc-Glu(OMe)-H (56 mg, 0.15 mmol) was dissolved in dimethylformamide (1 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazoro[4,5 -b] Pyridinium 3-oxide hexafluorophosphate (56 mg, 0.15 mmol) and N, N-diisopropylethylamine (0.046 mL, 0.27 mmol) were added, and stirred at room temperature for 1 minute. To the reaction mixture, a DMF solution (1 mL) of the known material Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) (150 mg, 0.13 mmol) was added and stirred for an additional 30 minutes. . After completion of the reaction, the reaction solution was diluted with a 1:1 = water: acetonitrile solution (containing 0.05 v/v% formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (14) (180 mg, 0.12 mmol).

MS(ESI)m/z: 1488.2[M+H] ⁺

Lithium hydroxide (8.7 mg, 0.36 mmol) and compound (14) (180 mg, 0.12 mmol) were dissolved in 1:1 = water: THF (8 mL) and stirred at room temperature for 15 minutes. After completion of the reaction, 1M aqueous hydrochloric acid solution was carefully added to the reaction solution to adjust the pH to 5 to 6. This was purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (15) (100 mg, 0.068 mmol).

MS(ESI)m/z: 1474.4[M+H] ⁺

Compound (15) (100 mg, 0.068 mmol) was dissolved in dimethylformamide (2 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazoro[4,5-b]pyri. Dinium 3-oxide hexafluorophosphate (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (0.035 mL, 0.20 mmol) were added, and stirred at room temperature for 1 minute. To the reaction mixture, N-(5-aminopentyl)maleimide hydrochloride (30 mg, 0.10 mmol) was added and stirred for an additional 5 minutes. After completion of the reaction, the reaction solution was diluted with a 1:1 = water: acetonitrile solution (containing 0.05 v/v% formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (16) (83 mg, 0.051 mmol).

MS(ESI)m/z: 1638.6[M+H] ⁺

Compound (16) (21 mg, 0.013 mmol) was dissolved in dimethylformamide (1 mL), diazabicycloundecene (0.0042 mL, 0.028 mmol) was added, and the mixture was stirred at room temperature for 2 minutes. After completion of the reaction, 1M aqueous hydrochloric acid solution was carefully added to the reaction solution to adjust the pH to 5 to 6. The reaction solution was diluted with a 1:1 = water: acetonitrile solution (containing 0.05 v/v% of formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (17) (10 mg, 0.0071 mmol).

MS(ESI)m/z: 1416.5[M+H] ⁺

Under an argon atmosphere, compound (17) (21 mg, 0.015 mmol) was dissolved in N,N-dimethylformamide (2 mL), 4-methylmorpholine (0.082 mL, 0.074 mmol), and succinic anhydride (7.4 mg, 0.074 mmol). mmol) and stirred at room temperature for 1 hour, the reaction solution was diluted with a 1:1 = water: acetonitrile solution (containing 0.05 v/v% of formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (12) (17.5 mg, 0.012 nmol).

¹ H NMR (500MHz, DMSO-d6) δ12.06 (br.s, 1H), 9.94-10.05 (m, 1H), 8.26-8.33 (m, 0.5H), 8.13-8.20 (m, 1H), 8.10 (d，J=7.8Hz，2H)，8.03-8.07(m，0.5H)，7.86-7.90(m，0.5H)，7.69-7.77(m，2H)，7.60-7.64(m，0.5H)， 7.55-7.60(m，2H)，7.29-7.35(m，3H)，7.23-7.29(m，3H)，7.17(s，1H)，6.99(s，2H)，5.94-6.02(m，1H)， 5.39-5.44(m，2H)，5.34(d，J=4.9Hz，1H)，4.94-5.12(m，2H)，4.70-4.77(m，1H)，4.58-4.68(m，1H)，4.49- 4.52(m，1H)，4.40-4.45(m，1H)，4.33-4.40(m，1H)，4.21-4.29(m，2H)，4.18-4.21(m，1H)，3.92-4.02(m，2H) )，3.76-3.80(m，1H)，3.53-3.61(m，1H)，3.41-3.50(m，1H)，3.36(t，J=7.1Hz，2H)，3.29-3.31(m，1H)， 3.21-3.26(m，4H)，3.20(s，2H)，3.17(s，2H)，3.11(s，2H)，2.90-3.07(m，5H)，2.82-2.89(m，3H)，2.31- 2.45(m，5H)，2.22-2.30(m，1H)，2.02-2.17(m，3H)，1.97-2.01(m，2H)，1.83-1.91(m，1H)，1.75-1.82(m，1H) )，1.65-1.75(m，2H)，1.50-1.62(m，2H)，1.42-1.50(m，4H)，1.26-1.39(m，3H)，1.13-1.22(m，2H)，0.96-1.06 (m，6H)，0.72-0.89(m，24H)

MS(ESI)m/z: 1516.4[M+H] ⁺

(2-3) Synthesis of Linker-payload(18)

Linker-Payload (18) was synthesized as follows.

Linker-payload (12) (10 mg, 0.0066 mmol) was dissolved in dimethylformamide (1 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazoro[4,5-b ]Pyridinium 3-oxide hexafluorophosphate (2.8 mg, 0.0073 mmol) and N,N-diisopropylethylamine (0.0023 mL, 0.013 mmol) were added, and stirred at room temperature for 1 minute. To the reaction mixture, methoxy-PEG8-amine (3.8 mg, 0.0099 mmol) was added and stirred for an additional 2 minutes. After completion of the reaction, the reaction solution was diluted with a 1:1 = water: acetonitrile solution (containing 0.05 v/v% formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (18) (12.1 mg, 0.064 mmol).

¹ H NMR (500MHz, DMSO-d6) δ9.89-10.00(m, 1H), 8.26-8.35(m, 1H), 8.10-8.15(m, 1H), 8.03-8.10(m, 1H), 7.92- 7.96(m，1H)，7.86-7.92(m，0.5H)，7.71-7.78(m，2H)，7.60-7.65(m，0.5H)，7.53-7.60(m，2H)，7.23-7.36(m) ，6H)，7.13-7.20(m，1H)，6.99(s，2H)，5.93-6.00(m，1H)，5.40-5.43(m，2H)，5.32-5.36(m，1H)，4.92-5.14 (m，2H)，4.69-4.78(m，1H)，4.58-4.68(m，1H)，4.45-4.51(m，1H)，4.39-4.45(m，1H)，4.31-4.39(m，1H) ，4.24-4.30(m，1H)，4.19-4.24(m，1H)，4.13-4.18(m，1H)，3.91-4.05(m，2H)，3.53-3.61(m，1H)，3.46-3.53( m，36H)，3.40-3.44(m，3H)，3.33-3.40(m，3H)，3.21-3.26(m，6H)，3.14-3.21(m，4H)，3.11(s，1H)，2.91- 3.06(m，4H)，2.82-2.90(m，2H)，2.22-2.44(m，6H)，2.05-2.16(m，3H)，1.94-2.04(m，2H)，1.84-1.93(m，1H) )，1.75-1.83(m，1H)，1.63-1.74(m，2H)，1.50-1.62(m，2H)，1.40-1.50(m，4H)，1.31-1.40(m，4H)，1.13-1.21 (m，2H)，0.95-1.07(m，6H)，0.71-0.90(m，24H)

MS(ESI)m/z: 1882.6[M+H] ⁺

(2-4) Synthesis of Linker-payload(19)

Linker-Payload (19) was synthesized as follows.

Maleimide (10) (8 mg, 0.0245 mmol) was dissolved in dimethylformamide (0.2 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazoro[4,5-b ]Pyridinium 3-oxide hexafluorophosphate (4.7 mg, 0.0123 mmol) and N,N-diisopropylethylamine (0.0085 mL, 0.0491 mmol) were added and stirred at room temperature for 5 minutes. For the reaction mixture, the known materials Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) (13.8mg, 0.0123mmol) and N,N-diisopropylethylamine (0.0086mL, 0.0491) mmol) was added and stirred for an additional 2 hours. After completion of the reaction, 1M aqueous hydrochloric acid solution was carefully added to the reaction solution to adjust the pH to 5 to 6. The reaction solution was diluted with a 1:1 = water: acetonitrile solution (containing 0.05 v/v% of formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (19) (12.1 mg, 0.064 mmol).

¹ H NMR (500MHz, DMSO-d6) δ9.98(brs, 1H), 9.77(brs, 1H), 8.40-8.27(m, 1H), 8.21(d, J=7.3Hz, 1H), 8.10-8.02 (m，1H)，7.88(d，J=8.8Hz，1H)，7.63-7.57(m，3H)，7.34-7.24(m，6H)，7.21-7.13(m，1H)，6.99(d，J =2.4Hz, 2H), 6.04-5.96(m, 1H), 5.46-5.40(m, 2H), 5.13-4.93(m, 4H), 4.79-4.60(m, 2H), 4.53-4.35(m, 3H) )，4.34-4.23(m，3H)，4.21-3.88(m，4H)，3.78(dd，J=9.5，2.2Hz，1H)，3.65-3.53(m，1H)，3.51-3.43(m，2H) )，3.40-3.34(m，2H)，3.28-3.16(m，8H)，3.14-3.10(m，2H)，3.08-2.81(m，5H)，2.41-1.90(m，8H)，1.87-1.64 (m，3H)，1.64-1.41(m，6H)，1.40-1.13(m，5H)，1.11-0.95(m，6H)，0.95-0.65(m，24H).

MS(ESI)m/z: 1431.2[M+H] ⁺

(2-5) Synthesis of Linker-payload(20)

Linker-Payload (20) was synthesized as follows.

Linker-payload (19) (14.6 mg, 0.0102 mmol) was dissolved in dimethylformamide (0.12 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazoro[4,5 -b] Pyridinium 3-oxide hexafluorophosphate (4.7 mg, 0.0122 mmol) and N, N-diisopropylethylamine (0.0035 mL, 0.0204 mmol) were added and stirred at room temperature for 5 minutes. To the reaction mixture, methoxy-PEG8-amine (4.7 mg, 0.0122 mmol) and N,N-diisopropylethylamine (0.0035 mL, 0.0204 mmol) were added and stirred for an additional 10 minutes. After completion of the reaction, 1M aqueous hydrochloric acid solution was carefully added to the reaction solution to adjust the pH to 5 to 6. The reaction solution was diluted with a 1:1 = water: acetonitrile solution (containing 0.05 v/v% of formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (20) (12.9 mg, 0.072 mmol).

MS(ESI)m/z: 1796.9[M+H] ⁺

(2-6) Synthesis of Linker-payload(21)

Linker-Payload (21) was synthesized as follows.

Linker-Payload (21) was synthesized according to the following scheme.

The MS analysis results of Linker-Payload (21) were as follows.

MS(ESI)m/z: 1544.8[M+H] ⁺

(2-7) Synthesis of Linker-payload (32)

Linker-Payload (32) was synthesized as follows.

Compound (17) (15 mg, 0.011 mmol) was dissolved in acrylic acid (1.5 mL) and stirred at 40°C for 24 hours, and the reaction solution was mixed with 1:1 = water: acetonitrile solution (0.05 v/v% of formic acid). (included) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (32) (2.5 mg, 0.0016 mmol).

¹ H NMR (500MHz, DMSO-d6) δ10.58(brs, 2H), 9.59(brs, 1H), 8.99-9.07(m, 1H), 7.98-8.04(m, 1H), 7.85-7.97(m, 2H)，7.72-7.78(m，1H)，7.59-7.68(m，2H)，7.22-7.36(m，8H)，7.11-7.20(m，1H)，6.99(s，2H)，6.80-6.93( m，1H)，5.49-5.89(m，4H)，4.91-5.14(m，2H)，4.69-4.78(m，1H)，4.57-4.67(m，1H)，4.35-4.53(m，3H)， 4.18-4.31(m，2H)，3.89-4.08(m，2H)，3.74-3.81(m，1H)，3.51-3.66(m，1H)，3.34-3.39(m，3H)，3.25-3.11(m ，10H)，2.91-3.07(m，5H)，2.84-2.90(m，3H)，2.65-2.83(m，4H)，2.37-2.43(m，5H)，2.22-2.33(m，1H)，2.09 -2.20(m，4H)，1.96-2.09(m，2H)，1.83-1.96(m，2H)，1.69-1.83(m，2H)，1.59-1.68(m，1H)，1.53-1.58(m， 1H)，1.42-1.52(m，4H)，1.26-1.40(m，3H)，1.14-1.25(m，2H)，0.94-1.06(m，6H)，0.71-0.89(m，24H)

MS(ESI)m/z: 1560.3[M+H] ⁺

(2-8) Synthesis of Linker-payload (33)

Linker-Payload (33) was synthesized as follows.

Imino diacetic acid (212 mg, 1.59 mmol) and sodium bicarbonate (534 mg, 6.36 mmol) were dissolved in water (4 mL). After foaming subsided, THF (2 mL) and Boc O (416 mg, 1.91 mmol) were added, and the mixture was stirred at room temperature for 72 hours. After removing THF by concentration under reduced pressure, the reaction solution was adjusted to about pH 1 in 1N hydrochloric acid. The aqueous layer was extracted with ethyl acetate (15 mLx3), the organic layer was dried with magnesium sulfate, and the filtered organic layer was concentrated to obtain compound (34) (205 mg, 0.877 mmol).

MS(ESI)m/z: 256.0[M+Na] ⁺

Compound (34) (14.6 mg, 0.0625 mmol), HATU (26.2 mg, 0.0689 mmol), and DIPEA (21.8 μL, 0.125 mmol) were dissolved in DMF (1.5 ml) and stirred at room temperature for 5 minutes. A solution of H2N-PEG8-OMe (52.7 mg, 0.138 mmol) and DIPEA (21.8 μL, 0.125 mmol) dissolved in DMF (1 mL) was added to the reaction solution, and stirred at room temperature for 1 hour. HATU (26.2 mg, 0.0689 mmol) and DIPEA (21.8 μL, 0.125 mmol) were added to the reaction solution, and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, diluted with a 1:1=water:acetonitrile solution, and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (35) (33.2 mg, 0.0344 mmol).

MS(ESI)m/z: 964.4[M+H] ⁺

Compound (35) (33.2 mg, 0.0344 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain compound (36) (33.6 mg, quant).

MS(ESI)m/z: 864.2[M+H] ⁺

Linker-payload (19) (6.6 mg, 0.0046 mmol), HATU (4.4 mg, 0.0046 mmol), and DIPEA (1.6 μL, 0.0092 mmol) were dissolved in DMF (0.15 ml) and stirred at room temperature for 5 minutes. A solution of compound (36) (5.0 mg, 0.0058 mmol) and DIPEA (1.6 μL, 0.0058 mmol) dissolved in DMF (0.15 mL) was added to the reaction solution, and stirred at room temperature for 3 hours. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (33) (2.0 mg, 0.00088 mmol).

MS(ESI)m/z: 2277.8[M+H] ⁺

(2-9) Synthesis of Linker-payload (37)

Linker-Payload (37) was synthesized as follows.

NHS-PEG9-NHS (164 mg, 0.231 mmol) was dissolved in DMF (1 mL). In another flask, compound (38) (35.5 mg, 0.112 mmol) was dissolved in DMF (1.5 mL), and this solution was added to the NHS-PEG9-NHS solution. The reaction solution was stirred at room temperature for 30 minutes, diluted with water (8 mL), and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (39) (56.8 mg, 0.0623 mmol).

MS(ESI)m/z: 912.2[M+H] ⁺

Fmoc-imino diacetic acid (13.6 mg, 0.0382 mmol), HATU (14.5 mg, 0.0382 mmol), and DIPEA (10.0 μL, 0.057 mmol) were dissolved in DMF (0.75 ml) and stirred at room temperature for 5 minutes. In another flask, a solution of H2N-VC-PAB-MMAE (42.9 mg, 0.0382 mmol) and DIPEA (10.0 μL, 0.057 mmol) dissolved in DMF (0.75 mL) was added to the Fmoc-imino diacetic acid solution, and incubated at room temperature. It was stirred for 18 hours. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (40) (21.2 mg, 0.0143 mmol).

MS(ESI)m/z: 1460.4[M+H] ⁺

Compound (40) (100 mg, 0.0695 mmol) was dissolved in DMF (2.5 mL), DBU (31 μL, 0.205 mmol) was added, and stirred at room temperature for 5 minutes. The reaction solution was diluted with a 1:1=water:acetonitrile solution (containing 0.05% formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (41) (86.6 mg, 0.0674 mmol).

MS(ESI)m/z: 1238.4[M+H] ⁺

Compound (39) (22.9 mg, 0.0185 mmol) and compound (41) (33.7 mg, 0.0178 mmol) were dissolved in 1:1 = water: acetonitrile solution (2 mL), and sodium bicarbonate (1.9 mg, 0.0222 mmol) was added. It was added and stirred at room temperature for 23 hours. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (37) (7.3 mg, 0.0036 mmol).

MS(ESI)m/z: 2035.5[M+H] ⁺

(2-10) Synthesis of Linker-payload (42)

Linker-Payload (42) was synthesized as follows.

Compound (39) (23.4 mg, 0.0257 mmol) and EVC-PAB-MMAE (21.4 mg, 0.0171 mmol) were dissolved in 1:1 = water: acetonitrile solution (3 mL), and sodium bicarbonate (10 mg, 0.119 mmol) was added. It was added and stirred at room temperature for 1 hour. The reaction solution was adjusted to about pH 4 with 1N hydrochloric acid, diluted with a 1:1=water:acetonitrile solution, and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (42) (16.4 mg, 0.008 mmol).

MS(ESI)m/z: 2049.8[M+H] ⁺

(2-11) Synthesis of Linker-payload (43)

Linker-Payload (43) was synthesized as follows.

Linker-payload (21) (15.6 mg, 0.074 mmol), HATU (5.4 mg, 0.014 mmol), and DIPEA (4.5 μL, 0.026 mmol) were dissolved in DMF (0.5 mL) and stirred at room temperature for 1 minute. m-PEG8-amine (BroadPharm #BP-21111) (7.5 mg, 0.0194 mmol) was added to the reaction solution and stirred at room temperature for 20 minutes. The reaction solution was diluted with a 1:1=water:acetonitrile solution (containing 0.05% formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (43) (5.2 mg, 0.0027 mmol).

MS(ESI)m/z: 955.2[M+2H] ²⁺

(1-4) Synthesis of Linker-payload mimic (44)

Linker-Payload mimic (44) was synthesized as follows.

Compound (45) (20.2 mg, 0.0293 mmol), HATU (13.4 mg, 0.0352 mmol), and DIPEA (6.0 μL, 0.035 mmol) were dissolved in DMF (0.3 mL) and stirred at room temperature for 1 minute. Compound (46) (5.4 mg, 0.035 mmol) was added and stirred at room temperature for 17 hours. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (47) (23.9 mg, quant).

MS(ESI)m/z: 817.4[M+H] ⁺

Compound (47) (23.9 mg, 0.0293 mmol) was dissolved in DMF (0.45 mL), then bis(4-nitrophenyl) carbonate (27.3 mg, 0.0879 mmol) and DIPEA (11.4 μL, 0.0657 mmol) were added, It was stirred at room temperature for 3 hours. Sarucosine-pyrene (44.3 mg, 0.147 mmol), HOBt (5.9 mg, 0.044 mmol), and DIPEA (39.4 μL, 0.227 mmol) were added to the reaction solution, and stirred at room temperature for 20 hours. Diethylamine (93.0 μL, 0.882 mmol) was added to the reaction solution, stirred at room temperature for 3 hours, and concentrated under reduced pressure. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (48) (1.9 mg, 0.0021 mmol).

MS(ESI)m/z: 923.5[M+H] ⁺

Compound (48) (1.9 mg, 0.0021 mmol) was dissolved in DMF (0.4 mL), then 4-maleimide hexanoic acid (0.7 mg, 0.003 mmol), HOAt (0.4 mg, 0.003 mmol), and WSC·HCl (0.9 mg, 0.005mmol), triethylamine (0.89μL, 0.0063mmol), and DMAP (0.1mg, 0.001mmol) were added, and stirred at room temperature for 5 hours. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (49) (0.3 mg, 0.0003 mmol).

MS(ESI)m/z: 1116.5[M+H] ⁺

Compound (49) (0.3 mg, 0.0003 mmol) was dissolved in 1,4-dioxane (0.3 mL), then 4N hydrogen chloride dioxane solution (315 μL, 1.26 mmol) was added, and the mixture was stirred at room temperature for 5 hours. The reaction solution was cooled to 0°C, DIPEA (238 μL, 1.39 mmol) was added, and stirred at room temperature for 1 minute. The reaction solution was concentrated, diluted with a 1:1=water:acetonitrile solution, and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (44) (0.3 mg, 0.0003 mmol).

MS(ESI)m/z: 1060.5[M+H] ⁺

(1-5) Synthesis of Linker-payload mimic (50)

Linker-Payload mimic (50) was synthesized as follows.

Compound (45) (24.8 mg, 0.0367 mmol), HATU (16.7 mg, 0.0440 mmol), and collidine (5.8 μL, 0.044 mmol) were suspended in acetonitrile (0.5 mL) and stirred at room temperature for 1 minute. Compound (51) (6.2 mg, 0.044 mmol) was added and stirred at room temperature for 24 hours. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (52) (11.4 mg, 0.0142 mmol).

MS(ESI)m/z: 805.4[M+H] ⁺

Compound (52) (11.4 mg, 0.0142 mmol) was dissolved in DMF (0.3 mL), then bis(4-nitrophenyl) carbonate (12.6 mg, 0.0426 mmol) and DIPEA (5.4 μL, 0.0318 mmol) were added. It was stirred at room temperature for 4 hours.

Bis(4-nitrophenyl) carbonate (6.5 mg, 0.0213 mmol) and DIPEA (2.7 μL, 0.016 mmol) were additionally added, and stirred at room temperature for 3 hours. Sarucosine-pyrene (42.9 mg, 0.142 mmol), HOBt (2.9 mg, 0.021 mmol), and DIPEA (38.4 μL, 0.226 mmol) were added to the reaction solution, and stirred at room temperature for 16 hours. Diethylamine (29.7 μL, 0.284 mmol) was added to the reaction solution, stirred at room temperature for 3 hours, and concentrated under reduced pressure. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (53) (10.0 mg, 0.0110 mmol).

MS(ESI)m/z: 911.4[M+H] ⁺

Compound (54) (10.0 mg, 0.0110 mmol) was dissolved in DMF (1 mL), then 4-maleimide hexanoic acid (3.5 mg, 0.017 mmol), HOAt (2.2 mg, 0.017 mmol), and WSC·HCl (4.8 mg). , 0.025 mmol), triethylamine (4.6 μL, 0.033 mmol), and DMAP (0.3 mg, 0.002 mmol) were added, and stirred at room temperature for 4 hours. The reaction solution was diluted with a 1:1=water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain compound (55) (1.6 mg, 0.0014 mmol).

MS(ESI)m/z: 1104.5[M+H] ⁺

Compound (55) (1.6 mg, 0.0014 mmol) was suspended in 1,4-dioxane (0.2 mL), then 4N hydrogen chloride dioxane solution (181 μL, 724 mmol) was added, and the mixture was stirred at room temperature for 5 hours. The reaction solution was cooled to 0°C, DIPEA (136 μL, 798 mmol) was added, and stirred at room temperature for 1 minute. The reaction solution was concentrated, diluted with a 1:1=water:acetonitrile solution, and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload (50) (0.81 mg, 0.00077 mmol).

MS(ESI)m/z: 1048.5[M+H] ⁺

Comparative Example 1: Synthesis of Linker-payload mimic (28)

Maleimide-VC-Pyrene (28) was synthesized as follows. It was synthesized in one step from commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) and known Sarcosine-pyrene (WO2018218004A1).

Commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) (15.5 mg, 0.021 mmol) was dissolved in dichloromethane (1 mL) and N,N-diisopropylethylamine (0.025 mL, 0.142 mL). mmol), a known dimethylformamide solution (0.5 mL) of Sarcosine-pyrene (WO2018218004A1) (7.6 mg, 0.025 mmol) was added, and stirred for 17 hours. After purification by reverse-phase preparative chromatography, the fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload mimic (28) (7.3 mg, 0.008 mmol). .

¹ H NMR (400MHz, DMSO-d6) δ9.98(s, 1H), 8.34(d, J=9.2Hz, 2H), 8.32-8.23(m, 4H), 8.16(s, 2H), 8.10-8.00 (m，4H)，7.80(d，J=8.8Hz，1H)，7.59(d，J=8.4Hz，2H)，7.31(d，J=8.0Hz，2H)，6.99(s，2H)，5.96 (m，1H)，5.40(s，2H)，5.01(s，2H)，4.95(d，J=6.0Hz，2H)，4.38(m，1H)，4.19(m，1H)，3.03-2.92( m，3H)，2.67(m，1H)，2.33(m，1H)，2.20-2.07(m，2H)，1.97(m，1H)，1.67(m，1H)，1.59(m，1H)，1.51 -1.45(m，6H)，1.26-1.15(m，3H)，0.83(dd，J=12.8，6.8Hz，6H)

MS(ESI)m/z: 901.45[M+H] ⁺

Comparative Example 2: Synthesis of Linker-payload

(2-1) Synthesis of Linker-Payload(29)

Linker-Payload (29) was synthesized as follows.

Linker-Payload (29) was synthesized according to the following scheme.

The MS analysis results of Linker-Payload (29) were as follows.

MS(ESI)m/z: 1447.8[M+H] ⁺

Example 3: Synthesis of ADC mimic

(3-1) Synthesis of ADC mimic

In the following comparative examples and examples, the antibody derivative (thiol group-introduced trastuzumab) described in Example 81-7 of International Publication No. 2019/240287 (WO2019/240287A1) was used as the thiol group-introduced antibody. The antibody derivative has the following structure in which a thiol group is position-selectively introduced into Trastuzumab (humanized IgG1 antibody) through the amino group of the side chain of the lysine residue at position 246 or 248 of the antibody heavy chain (the position of the lysine residue is according to EU numbering).

(In the above structure, NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ - extending from the antibody heavy chain corresponds to the side chain of the lysine residue, and HS-CH ₂ which is a thiol-containing group with respect to the amino group in the side chain of the lysine residue -CH ₂ -C(=O) is added.)

Add 10 equivalents of the DMF solution (1.25mM) of the Linker-payload mimic synthesized in Comparative Example 1 and Example 1 to the buffer (pH 7.4 PBS buffer) solution (20 μM) of the thiol group-introduced antibody, and leave for 2 hours at room temperature. , ADC mimic was obtained by purification using NAP-5 Columns (manufactured by GE Healthcare).

ADC mimic 1 with the following structure was synthesized from the Linker-payload mimic (1) synthesized in Example 1-1 and the thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 150641 in which two Linker-payload mimics (1) were introduced.

Similarly, ADC mimic 2 with the following structure was synthesized from the Linker-payload mimic (5) of Example 1-2 and a thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 150803 in which two Linker-payload mimics (5) were introduced.

Similarly, ADC mimic 3 with the following structure was synthesized from the Linker-payload mimic (9) of Example 1-3 and a thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 150620 in which two Linker-payload mimics (9) were introduced.

Similarly, ADC mimic 4 with the following structure was synthesized from the Linker-payload mimic (28) of Comparative Example 1 and a thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 150244 where two Linker-payload mimics (28) were introduced.

(3-2) DAR analysis of ADC mimic

ESI-TOFMS analysis of the ADC mimic synthesized in Example 3-1 was performed according to the report (WO2019/240287A1), and the DAR was confirmed to be 2.

Example 4: Synthesis of ADC

(4-1) Synthesis of ADC

Add 10 equivalents of the DMF solution (1.25mM) of the Linker-payload mimic synthesized in Comparative Example 2 and Example 1 to the buffer (pH 7.4 PBS buffer) solution (20μM) of the thiol group-introduced antibody, and leave at room temperature for 2 hours. , ADC was obtained by purification using NAP-5 Columns (manufactured by GE Healthcare).

ADC1 of the following structure was synthesized from the Linker-payload (11) synthesized in Example 2-1 and the thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 151230 where two Linker-payloads (11) were introduced.

ADC2 of the following structure was synthesized from the Linker-payload (12) synthesized in Example 2-2 and the thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 151443 where two Linker-payloads (12) were introduced.

ADC3 with the following structure was synthesized from the Linker-payload (18) synthesized in Example 2-3 and the thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 152173 where two Linker-payloads (18) were introduced.

ADC4 of the following structure was synthesized from the Linker-payload (19) synthesized in Example 2-4 and the thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 151270 where two Linker-payloads (19) were introduced.

ADC5 of the following structure was synthesized from the Linker-payload (20) synthesized in Example 2-5 and the thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 152005 where two Linker-payloads (20) were introduced.

ADC6 of the following structure was synthesized from the Linker-payload (21) synthesized in Example 2-6 and the thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 151492 where two Linker-payloads (21) were introduced.

ADC7 of the following structure was synthesized from the Linker-payload (29) synthesized in Comparative Example 2 and the thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 151295 where two Linker-payloads (29) were introduced.

ADC 8 with the following structure was synthesized from commercially available MC-VC-MMAE (CAS no: 646502-53-6) and a thiol-containing antibody. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 151091 where two MC-VC-MMAE were introduced.

Similarly, ADC mimic 9 with the following structure was synthesized from the Linker-payload (32) and thiol-containing antibody of Example 2-7. By performing ESI-TOFMS analysis, the reaction product was confirmed to have a peak at 152261 where two Linker-payloads (32) were introduced.

(4-2) DAR analysis of ADC

ESI-TOFMS analysis of the ADC synthesized in Example 4-1 was performed according to the report (WO2019/240287A1), and it was confirmed that the DAR was 2.

Example 5: Evaluation of hydrophobicity of ADC and ADC mimic by hydrophobic column chromatography (HIC-HPLC)

HIC-HPLC analysis was performed according to the report (Anal. Chem., 2019, 91, 20, 12724-12732). Measurements were performed using the following conditions. The hydrophobicity of ADC can be evaluated by the retention time of ADC in the HIC chromatogram.

Measurement system: Chromaster (registered trademark) (manufactured by Hitachi)

Column: Tosoh Biobuthyl NPR 2.5μm 4.6×35mm column manufactured by Tosoh Bioscience.

Gradient: Linear gradient of eluent A/B

Flow rate: 0.8mL/min

Eluent A: 1.1M (NH ₄ ) ₂ SO ₄ ，25mM Na ₂ HPO ₄ /NaH ₂ PO ₄ (pH 6.0)

Eluent B: 25mM Na ₂ HPO ₄ /NaH ₂ PO ₄ (pH 6.0, 25v/v% isopropanol added)

Detector: UV (280nm)

Example 6: Evaluation of the aggregation rate of ADC and ADC mimic by size exclusion chromatography (SEC-HPLC)

SEC-HPLC analysis was performed according to the report (ChemistrySelect, 2020, 5, 8435-8439). Measurements were performed using the following conditions.

Measurement system: 1260 HPLC system (manufactured by Agilent)

Column: AdvanceBio SEC 300Å 2.7μm, 4.6mm×150mm manufactured by Agilent.

Flow rate: 0.25mL/min

Eluent: 100mM sodium dihydrogen phosphate/sodium hydrogen phosphate, 250mM aqueous solution of sodium chloride (pH 6.8), 10%v/v isopropanol.

Detector: UV (280nm)

Example 7: Evaluation of ADC mimic using enzyme cathepsin B

The cleavage ability of various ADC mimics by cathepsin B was evaluated by analyzing the amount of fluorescent molecules shed from the ADC mimics as follows.

(7-1) Cathepsin B cleavage test

According to the notation (Nature Communications 2018, 9, 2512), it was carried out as follows. To 180 μL of MES buffer (10mM MES, 40 μM DTT, pH 5.0), ADC mimic was added to a concentration of 1 mg/mL, and then 30 μL was dispensed into six Eppen tubes. For three of the six samples, 100 μL of acetonitrile was immediately added at 0°C, stirred with a vortex, and then centrifuged to obtain precipitates. The generated supernatant solution was recovered and subjected to HPLC analysis. The remaining three were incubated at 37°C for 6 hours. 100 μL of acetonitrile was added to each sample, stirred by vortex, and then centrifuged to obtain a precipitate. The resulting supernatant solution was recovered and subjected to HPLC analysis.

(7-2) Analysis of the amount of shed fluorescent molecules using HPLC analysis

For measurement, the fluorescent molecular weight shed from the ADC mimic was measured using liquid chromatography/fluorescence detection method. In Example 7-1, three samples to which acetonitrile was immediately added at 0°C were taken as 0 hours, and three samples incubated at 37°C for 6 hours as described in Example 7-1 were taken as 6 hours. And the difference in luminescence intensity between the 6-hour sample and the 0-hour sample was analyzed.

Pyrene was used separately to calculate the correlation between the area area of fluorescence intensity by HPLC and concentration. Using the above calculation formula, the difference in luminescence intensity of each ADC mimic was converted into concentration. The ratio of the difference in fluorescence intensity when the concentration at time 0 was 100% was calculated as the dropout rate.

As a result, the ADC mimic synthesized in Example 1 had high reactivity to cathepsin and immediately released fluorescent molecules, whereas the ADC mimic synthesized in Comparative Examples 1-1 and 1-2 had low reactivity to cathepsin. .

Example 8: Evaluation of ADC using the enzyme cathepsin B

The cleavage ability of various ADCs by cathepsin B was evaluated by analyzing the amount of fluorescent molecules eliminated from the ADCs as follows.

(8-1) Cathepsin B cleavage test

This study was conducted according to the published report (Nature Communications 2018, 9, 2512).

(8-2) Analysis of the amount of shed payload using HPLC analysis

The measurement used liquid chromatography mass spectrometry (including tandem mass spectrometry) to measure the amount of payload dropped from the ADC. In Example 8-1, three samples to which acetonitrile was immediately added at 0°C were taken as 0 hours, and in Example 8-1, three samples incubated at 37°C for 6 hours were taken as 6 hours. The MS intensities of the payload detected from the sample and the 0-hour sample were respectively calculated using extracted ion chromatograms, and these differences were analyzed.

Separately, MMAE was used to calculate the correlation between the area area and concentration of TIC by HPLC. The TIC of the fluorescence intensity of each ADC was converted to concentration using the above calculation formula. The ratio of the difference in the ion chromatogram when the concentration on Day 0 was 100% was calculated as the dropout rate.

As a result, it was found that the ADC mimic synthesized in Example 2 had high reactivity to cathepsin and immediately released the payload.

Example 9: Evaluation of ADC mimic using mouse plasma

(9-1) Stability test of ADC mimic in plasma

To 500 μL of mouse plasma (manufactured by Charles River), ADC mimic was added to a concentration of 0.1 mg/mL and then sterilized and filtered. 50 μL of the solution was dispensed into six Eppen tubes. Three of the six samples were stored in an incubator set at 37°C for 4 days. The remaining three were stored in the same freezer at -80°C for 4 days. 100 μL of acetonitrile was added to each sample, stirred by vortex, and then centrifuged to obtain a precipitate. The resulting supernatant solution was recovered and subjected to HPLC analysis.

(9-2) Analysis of the amount of shed fluorescent molecules using HPLC analysis

For measurement, the fluorescent molecular weight shed from the ADC mimic was measured using liquid chromatography/fluorescence detection method. The three samples stored in the freezer in Example 9-1 were set as Day = 0, and the three samples stored at 37°C in Example 9-1 were set as Day = 4. Luminescence at Day = 4 and Day = 0 The difference in intensity was interpreted.

Calculation of the dropout rate of fluorescent molecules was performed according to Example 7-2.

The results evaluated the dropout rate of fluorescent molecules as shown in the table below.

Example 10: Evaluation of ADC using mouse plasma

(10-1) Stability test of ADC in plasma

To 500 μL of mouse plasma (manufactured by Charles River), ADC mimic was added to a concentration of 0.1 mg/mL and then sterilized and filtered. 50 μL of the solution was dispensed into six Eppen tubes. Three of the six samples were stored in an incubator set at 37°C for 4 days. The remaining three were stored in the same freezer at -80°C for 4 days. 100 μL of acetonitrile was added to each sample, stirred by vortex, and then centrifuged to obtain a precipitate. The resulting supernatant solution was recovered and subjected to HPLC analysis.

(10-2) Analysis of the amount of dropped payload using HPLC analysis

The measurement used liquid chromatography mass spectrometry (including tandem mass spectrometry) to measure the amount of payload dropped from the ADC. In Example 10-1, three samples to which acetonitrile was immediately added at 0°C were regarded as 0 hours, and in Example 8-1, three samples incubated at 37°C for 6 hours were regarded as 6 hours. The MS intensities of the payload detected from the sample and the 0-hour sample were respectively calculated using extracted ion chromatograms, and these differences were analyzed. The payload drop rate was calculated according to Example 8-2.

Claims (20)

Formula (I):
[Formula (I)]

〔During the meal,
Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and also forms an amide bond with a carbonyl group adjacent to Ig regioselectively through the amino group in the side chain of the lysine residue in the two heavy chains,
L ₁ and L ₂ each represent a divalent group,
R ₁ represents a monovalent group that may contain a hydrophilic group,
X shows a good two -oriented group even if you have a substituent, and where the secondary group is 1 to 3, which constitutes the main chain that connects the two atoms on both sides of the X, and even if the substituent contains a hydrophilic group. Good monovalent energy,
D represents a functional material,
R _A represents the side chain of the valine residue,
R _B represents the side chain of a citrulline residue or an alanine residue,
n is 0 or 1, and here, when n is ¹ , the substituent on
The average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5. A conjugate or salt thereof of an antibody and a functional substance, comprising a structural unit represented by <RTI ID=0.0>and</RTI> and at least one hydrophilic group is present in the structural unit. . The conjugate or salt thereof according to claim 1, wherein the immunoglobulin unit is a human immunoglobulin unit. The conjugate or salt thereof according to claim 2, wherein the human immunoglobulin unit is a human IgG antibody. The conjugate or salt thereof according to claim 1, wherein the lysine residue is present at position 246/248, position 288/290 or position 317 according to Eu numbering. The conjugate or salt thereof according to claim 1, wherein r is 1.9 to 2.1. The conjugate or salt thereof according to claim 1, wherein the hydrophilic group is at least one group selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety. The method of claim 1, wherein L ₁ has the following formula (i):
[Formula (i)]
-L ₄ -Y L ₃ -
〔During the meal,
L ₃ and L ₄ are each independently, -(C(R) ₂ ) _m -, -(OC(R) ₂ -C(R) ₂ ) _m - and -(C(R) ₂ -C(R) ₂ -O) _m - and a divalent group selected from the group consisting of combinations thereof,
R is each independently a hydrogen atom, alkyl with 1 to 6 carbon atoms, alkenyl with 2 to 6 carbon atoms, or alkynyl with 2 to 6 carbon atoms,
m is an integer from 0 to 20,
Y is a divalent group produced by the reaction of two bioorthogonal functional groups capable of reacting with each other.] A conjugate or a salt thereof, which represents a divalent group represented by. The conjugate or salt thereof according to claim 7, wherein L ₃ and L ₄ each independently include -(C(R) ₂ ) _m -. The method of claim 7, wherein Y has the following structural formula:

[Here, white circles and black circles represent bonding hands,
If the bond in the white circle is bonded to L ₃ , the bond in the black circle is bonded to L ₄ ,
When the bond in the white circle is bonded to L ₄ , the bond in the black circle is bonded to L _3. ] A divalent group, conjugate, or salt thereof represented by any of the following structural formulas. The conjugate according to claim 1, wherein the structural unit represented by formula (I) is a structural unit represented by the following formula (I-1), (I-2), (I-3), or (I-4). or salts thereof:
[Formula (I-1)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
R ₁ represents a monovalent group containing a hydrophilic group.];
[Formula (I-2)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
R ₂ represents a monovalent group containing a hydrophilic group.];
[Formula (I-3)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
R ₃ represents a hydrogen atom, an alkyl having 1 to 6 carbon atoms, or a monovalent group containing a hydrophilic group,
R ₄ represents a monovalent group containing a hydrophilic group.] or
[Formula (I-4)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
R ₁ represents a hydrogen atom or alkyl having 1 to 6 carbon atoms,
R ₆ represents a monovalent group containing a hydrophilic group.]. The conjugate or salt thereof according to claim 10, wherein the structural unit represented by formula (I) is a structural unit represented by formula (I-1), (I-2), or (I-3). The method of claim 10, wherein the structural unit represented by formula (I-1), (I-2), (I-3) or (I-4) has the following formula (I-1a'), (I-1b') ), (I-1c'), (I-2'), (I-3a'), (I-3b'), (I-4a'), (I-4b') or (I-4c') A conjugate or a salt thereof, which is a structural unit represented by:
[Formula (I-1a')]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group.];
[Formula (I-1b')]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
Two L ₅ each independently represent a bond or a divalent group,
Two HGs each independently represent a hydrophilic group.];
[Formula (I-1c')]

〔During the meal,
Ig, L ₂ , D, R _A , R _B , r are the same as in formula (I),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group,
L _1a and L _1b each independently represent a bond or a divalent group,
HG' represents a divalent hydrophilic group.];
[Formula (I-2')]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group.];
[Formula (I-3a')]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group.];
[Formula (I-3b')]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
HG represents a hydrophilic group.];
[Formula (I-4a')]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group.];
[Formula (I-4b')]

〔During the meal,
Ig, L ₂ , D, R _A , R _B , r are the same as in formula (I),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group,
L _1a and L _1b each independently represent a bond or a divalent group,
HG' represents a divalent hydrophilic group.] or
[Formula (I-4c')]

〔During the meal,
Ig, L ₁ , D, R _A , R _B , r are the same as in formula (I),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group,
L ₂ ' represents a bond or divalent group,
E represents an electron-withdrawing group,
n2 is an integer from 1 to 4.]. 13. The method of claim 12, wherein the formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), (I-3b'), ( The structural unit represented by I-4a'), (I-4b') or (I-4c') has the following formula (I-1a'-1), (I-1a'-2), (I-1b') -1), (I-1c'-1), (I-2'-1), (I-2'-2), (I-3a'-1), (I-3a'-2), ( A structural unit represented by I-3b'-1), (I-4a'-1), (I-4b'-1), (I-4c'-1) or (I-4c'-2), Conjugate or salt thereof:
[Formula (I-1a'-1)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-1a'-2)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-1b'-1)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-1c'-1)]

〔During the meal,
Ig, L ₂ , D, R _A , R _B , r are the same as in formula (I),
L _1a and L _1b each independently represent a bond or a divalent group,
n1 is an integer from 3 to 20.];
[Formula (I-2'-1)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-2'-2)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-3a'-1)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-3a'-2)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-3b'-1)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-4a'-1)]

〔During the meal,
Ig, L ₁ , L ₂ , D, R _A , R _B , r are the same as in formula (I)];
[Formula (I-4b'-1)]

〔During the meal,
Ig, L ₂ , D, R _A , R _B , r are the same as in formula (I),
L _1a and L _1b each independently represent a bond or a divalent group,
n1 represents an integer from 3 to 20.];
[Formula (I-4c'-1)]

〔During the meal,
Ig, L ₁ , D, R _A , R _B , r are the same as in formula (I),
L ₂ ' represents a bond or a divalent group.] or
[Chemical formula (I-4c'-2)]

〔During the meal,
Ig, L ₁ , D, R _A , R _B , r are the same as in formula (I),
L ₂ ' represents a bond or a divalent group.] The method according to any one of claims 1 to 13, wherein L ₂ has the following structural formula:

(Here, black circles and white circles represent bonding hands,
The binding hand in the black circle is bonded to the carbonyl group adjacent to L ₂ ,
The binding hand in the white circle is connected to D,
E represents an electron-withdrawing group,
n2 is an integer of 1 to 4.) A conjugate or a salt thereof containing a divalent group represented by . Formula (II-1), (II-2), (II-3), (II-4b') or (II-4c'):
[Formula (II-1)]

〔During the meal,
L ₂ and L ₃ each represent a divalent group,
R ₁ represents a monovalent group that may contain a hydrophilic group,
D represents a functional material,
R _A represents the side chain of the valine residue,
R _B represents the side chain of a citrulline residue or an alanine residue,
B represents a bioorthogonal functional group];
[Formula (II-2)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
R ₂ represents a monovalent group that may contain a hydrophilic group.];
[Formula (II-3)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
R ₃ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or a monovalent group containing a hydrophilic group,
R ₄ represents a monovalent group containing a hydrophilic group.];
[Formula (II-4b')]

〔During the meal,
L ₂ , D, R _A , R _B and B are the same as in formula (II-1),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group,
L _3a and L _3b each independently represent a bond or a divalent group,
HG' represents a divalent hydrophilic group.] or
[Formula (II-4c')]

〔During the meal,
L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group,
L ₂ ' represents a bond or divalent group,
E represents an electron-withdrawing group,
n2 is an integer of 1 to 4.], a compound or a salt thereof. The compound or salt thereof according to claim 15, wherein the bioorthogonal functional group is a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. The method of claim 15, wherein the compound represented by formula (II-1), (II-2) or (II-3) has the following formula (II-1a'), (II-1b'), (II-1c') ), (II-2'), (II-3a') or (II-3b'), a compound or a salt thereof:
[Formula (II-1a')]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group.];
[Formula (II-1b')]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
Two L ₅ each independently represent a bond or a divalent group,
Two HGs each independently represent a hydrophilic group.];
[Formula (II-1c')]

〔During the meal,
L ₂ , D, R _A , R _B and B are the same as in formula (II-1),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group,
L _3a and L _3b each independently represent a bond or a divalent group,
HG' represents a divalent hydrophilic group.];
[Formula (II-2')]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group.];
[Formula (II-3a')]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
L ₅ represents a bond or a divalent group,
HG represents a hydrophilic group.] or
[Formula (II-3b')]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
HG represents a hydrophilic group.]. 16. The method of claim 15, wherein the formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a'), (II-3b'), ( Compounds represented by (II-4b') or (II-4c') have the following formulas (II-1a'-1), (II-1a'-2), (II-1b'-1), (II-1c) '-1), (II-2'-1), (II-2'-2), (II-3a'-1), (II-3a'-2), (II-3b'-1), Compound or salt thereof, represented by (II-4b'-1), (II-4c'-1) or (II-4c'-2):
[Formula (II-1a'-1)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1)];
[Formula (II-1a'-2)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1)];
[Formula (II-1b'-1)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1)];
[Formula (II-1c'-1)]

〔During the meal,
L ₂ , D, R _A , R _B and B are the same as in formula (II-1),
L _3a and L _3b each independently represent a bond or a divalent group,
n1 is an integer from 3 to 20.];
[Formula (II-2'-1)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1)];
[Formula (II-2'-2)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1)];
[Formula (II-3a'-1)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1)];
[Formula (II-3a'-2)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1)] or
[Formula (II-1b'-1)]

〔During the meal,
L ₂ , L ₃ , D, R _A , R _B and B are the same as in formula (II-1)];
[Formula (II-4b'-1)]

〔During the meal,
L ₂ , D, R _A , R _B and B are the same as in formula (II-1),
L _3a and L _3b each independently represent a bond or a divalent group,
n1 is an integer from 3 to 20.];
[Formula (II-4c'-1)]

〔During the meal,
L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
L ₂ ' represents a bond or a divalent group.] or
[Formula (II-4c'-2)]

〔During the meal,
L ₃ , D, R _A , R _B and B are the same as in formula (II-1),
L ₂ 'represents a bond or a divalent group.〕. The method of claim 15, wherein L ₂ has the following structural formula:

(Here, black circles and white circles represent bonding hands,
The binding hand in the black circle is bonded to the carbonyl group adjacent to L ₂ ,
The binding hand in the white circle is connected to D,
E represents an electron-withdrawing group,
n2 is an integer of 1 to 4.) A compound or salt thereof containing a divalent group represented by . A reagent for derivatizing an antibody, comprising the compound or a salt thereof according to any one of claims 15 to 19.